Reversibly crosslinked micelle systems

ABSTRACT

The present invention provides amphiphilic telodendrimers that aggregate to form nanocarriers characterized by a hydrophobic core and a hydrophilic exterior. The nanocarrier core may include amphiphilic functionality such as cholic acid or cholic acid derivatives, and the exterior may include branched or linear poly(ethylene glycol) segments. Nanocarrier cargo such as hydrophobic drugs and other materials may be sequester in the core via non-covalent means or may be covalently bound to the telodendrimer building blocks. Telodendrimer structure may be tailored to alter loading properties, interactions with materials such as biological membranes, and other characteristics.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Nos.61/485,774, filed May 13, 2011, and 61/487,953, filed May 19, 2011,which are incorporated in their entirety herein for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant Nos.CA115483 and CA 140449 awarded by the National Institutes of Health. TheGovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

Several effective chemotherapeutic agents for treatment of variouscancer types are very insoluble in water, requiring formulations thatinduce unwanted side effects. Recently, nanotherapeutic formulationssuch as Abraxane® (paclitaxel-loaded albumin nanoparticles), Doxil®(doxorubicin-loaded liposomes), and others have been shown to improvethe clinical toxicity profiles of the drugs, but their anti-tumoreffects are only marginally better than the original drug formulations.This has been attributed in part to the relatively large size of thenanotherapeutic formulations (generally >100 nm), which limits theextent to which the drugs can penetrate into tumor mass. In some cases,this large size also causes nanotherapeutics to be trapped in the liverand reticuloendothelial system (RES). Accordingly, there is a need todevelop smaller (20-80 nm) stealth and biocompatible nanocarriers foreffective delivery anti-cancer drugs in vivo.

We have recently developed several novel nanocarriers for paclitaxel(PTX) or other hydrophobic drugs. These novel nanocarriers, comprisingpoly(ethylene glycol) (PEG) and oligo-cholic acids, can self-assembleunder aqueous conditions to form core-shell (cholane-PEG) structuresthat can carry PTX in the hydrophobic interior. These amphiphilicdrug-loaded nanoparticles are therapeutic by themselves with improvedclinical toxicity profiles. More importantly, when decorated with cancercell surface targeting ligands and/or tumor blood vessel ligands, thesenanocarriers will be able to deliver toxic therapeutic agents to thetumor sites. The final size of the nanocarriers (10 to 100 nm) istunable by using various, or a combination of, different cholane-PEGpreparations. The nanocarrier components, PEG and cholic acid, are allbiocompatible and largely non-toxic. Indeed, the PTX nanotherapeuticsexhibited safe profile in in vivo administration for anticancertreatment in mouse models and companion dogs. However, some nanocarriersexhibited some hemolytic activity both in vitro and in vivo, as well asreduced stability and loading capacity for certain drugs. Therefore,there is a need to develop nanocarriers with improved stability,biocompatibility and versatility.

The present invention is based on the surprising discovery that certaincrosslinkable functional groups can be introduced into telodendrimers,therefore crosslinking the nanoparticles reversibly to minimizepremature drug release and increase in vitro and in vivo stability ofthe nanotherapeutics. The crosslinked nanotherapeutics improve thetherapeutic properties without disrupting nanocarrier assembly and drugloading capacity and stability, therefore addressing the needs describedabove.

BRIEF SUMMARY OF THE INVENTION

In some embodiments, the invention provide a compound of formula I:

(PEG)_(m)-A(Y¹)_(p)-L-D(Y²)_(q)—(R)_(n)  (I)

wherein radical A of formula I is linked to at least one PEG group.Radical D of formula I is a dendritic polymer having a single focalpoint group, a plurality of branched monomer units X and a plurality ofend groups. Radical L of formula I is a bond or a linker linked to thefocal point group of the dendritic polymer. Each PEG of formula I is apolyethyleneglycol (PEG) polymer, wherein each PEG polymer has amolecular weight of 1-100 kDa. Each R of formula I can be the end groupof the dendritic polymer, a hydrophobic group, a hydrophilic group, anamphiphilic compound or a drug, such that when R is not an end groupthen each R is linked to one of the end groups. Each Y¹ and Y² offormula I is a crosslinkable group that can be any of boronic acid,dihydroxybenzene or a thiol. Subscript m of formula I is an integer from0 to 20. Subscript n of formula I is an integer from 2 to 20, whereinsubscript n is equal to the number of end groups on the dendriticpolymer, and wherein at least half the number n of R groups that caneach be a hydrophobic group, a hydrophilic group, an amphiphiliccompound or a drug. And, each of subscripts p and q are 0 or from 2 to8, such that one of subscripts p and q is from 2 to 8.

In some embodiments, the invention provides a nanocarrier having aninterior and an exterior, the nanocarrier including at least twoconjugates, wherein each conjugate includes a polyethylene glycol (PEG)polymer, at least two amphiphilic compounds having both a hydrophilicface and a hydrophobic face, at least two crosslinking groups, and adendritic polymer covalently attached to the PEG, the amphiphiliccompounds and the crosslinking groups, wherein each conjugateself-assembles in an aqueous solvent to form the nanocarrier such that ahydrophobic pocket is formed in the interior of the nanocarrier, whereinthe PEG of each compound self-assembles on the exterior of thenanocarrier, and wherein at least two conjugates are reversiblycrosslinked via the crosslinking groups.

In some embodiments, the present invention provides a method ofreversing the cross-linking of the reversibly crosslinked nanocarrier ofthe present invention, by contacting the reversibly crosslinkednanocarrier with a bond cleavage component suitable for cleaving thecross-linked bond, thereby reversing the cross-linking of the reversiblycrosslinked nanocarrier.

In some embodiments, the present invention provides a method of treatinga disease, including administering to a subject in need of suchtreatment, a therapeutically effective amount of a nanocarrier of thepresent invention, wherein the nanocarrier includes a drug. The drug canbe a covalently attached to a conjugate of the nanocarrier.

In some embodiments, the present invention provides a method ofdelivering a drug to a subject in need thereof by administering ananocarrier of the present invention to the subject, wherein thenanocarrier includes the drug and a plurality of cross-linked bonds. Themethod also includes cleaving the cross-linked bonds using a bondcleavage component, such that the drug is released from the nanocarrier,thereby delivering the drug to the subject.

In some embodiments, the present invention provides a method of imaging,including administering to a subject to be imaged, an effective amountof a nanocarrier of the present invention, wherein the nanocarrierincludes an imaging agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic presentation of the disulfide cross-linkedmicelles formed by oxidation of thiolated telodendrimerPEG^(5k)-Cys₄-L₈-CA_(o) after self-assembly.

FIG. 2 shows the absorbance of PEG^(5k)-Cys₄-L₈-CA₈ micelle solutions inEllman's test (A) and the thiol conversions (B) as a function ofoxidation time. PTX loading (C) and size change (D) ofPEG^(5k)-Cys₄-L₈-CA₈ micelle before and after cross-linking versus thelevel of drug added at initial loading. The volume of the final micellesolution was kept at 1 mL and the final concentration of the polymers at20 mg/mL. DLS size distribution (E) and TEM image (F) of PTX-loadedcross-linked micelles (PTX loading was 4.6 mg/mL, TEM scale bar: 50 nm).

FIG. 3 shows the particle size of PTX loaded non-crosslinked micelles(NCMs) (PTX loading: 5.0 mg/mL) in human plasma 50% (v/v) for 1 min (A),24 h (B) and PTX loaded disulfide crosslinked micelles (DCMs) (PTXloading: 4.6 mg/mL) in plasma 50% (v/v) for 1 min (C) and 24 h (D) at37° C., respectively. The particle size of re-hydrated PTX-DCMs fromlyophilized PTX-DCMs powder in the absence (E) and in the presence (F)of 2.5 mg/mL SDS.

FIG. 4 shows (A) the stability in particle size of NCMs and DCMs in thepresence of 2.5 mg/mL SDS measured by DLS. TEM images of NCMs (B), DCMs(C) and DCMs treated with 10 mM GSH for 30 min (D) in the presence of2.5 mg/mL SDS (scale bar: 50 nm).

FIG. 5 shows (A) PTX release profiles of DCMs at different GSHconcentrations. GSH-responsive PTX release profiles of fresh preparedPTX-DCMs (B) and re-hydrated lyophilized PTX-DCMs (C) by adding GSH (10mM) at a specific release time (5 h) comparing with PTX-NCMs.NAC-responsive PTX release profiles of PTX-DCMs (D) by adding NAC (10mM) at a specific release time (5 h). Values reported are the meandiameter±SD for triplicate samples.

FIG. 6 shows MTT assays showing the viability of SKOV-3 cells after 2 hincubation with (A) different concentrations of empty NCMs and DCMs; and(B) Taxol®, PTX-NCMs and PTX-DCMs with and without pre-treatment of 20mM GSH-OEt. (C) In vitro red blood cell (RBC) lysis of empty NCMs andDCMs. Values reported are the mean±SD for triplicate samples.

FIG. 7 shows the fluorescence signal of BODIPY labeled (A) and DiDloaded (B) DCMs and NCMs in the blood collected at different time pointsafter i.v. injection in the nude mice.

FIG. 8 shows in vivo and ex vivo near infra-red fluorescence (NIRF)optical imaging. Top: In vivo NIRF optical images of SKOV-3 xenograftbearing mouse were obtained with Kodak imaging system at different timepoints after i.v. injection of DCMs co-loaded with PTX and DiD; Bottom:Ex vivo NIR image of dissected organs and tumor was obtained at 72 hafter injection.

FIG. 9 shows (A) In vivo anti-tumor efficacy after intravenous treatmentof different PTX formulations in the subcutaneous mouse model of SKOV-3ovarian cancer. Tumor bearing mice were administered i.v. with PBS(control) and different PTX formulations on days 0, 3, 6, 9, 12 and 15when tumor volume reached about 100-200 mm³ (n=8-10). (B) Survival curveof mice in different treatment groups. (C) Complete tumor response rateof the three groups of mice treated with total six doses of PTX micellarformulations at the dose of 30 mg/kg.

FIG. 10 shows the chemical structure (A) and schematic representation(B) of PEG^(5k)-Cys₄-L₈-CA₈.

FIG. 11 shows the MALDI-TOF MS of PEG^(5k)-Cys₄-L₈-CA₈ telodendrimercomparing with the starting PEG 5000 and PEG^(5k)-CA₈ telodendrimer.

FIG. 12 shows ¹H NMR spectra of PEG^(5k)-Cys₄-L₈-CA₈ telodendrimerrecorded in CDCl₃ and D₂O.

FIG. 13 shows the particle size of NCMs in the absence (A) and in thepresence (B) of 2.5 mg/mL SDS for 10 sec. The particle size of DCMs inthe absence (C) and in the presence of 2.5 mg/mL SDS (D), 2.5 mg/mLSDS+2 μM GSH (E), 2.5 mg/mL SDS+10 mM GSH (F) for 40 min. The particlesize was measured by dynamic light scattering (Microtrac).

FIG. 14 shows cumulative PTX release profile from Taxol®, PTX loadedNCMs and DCMs.

FIG. 15 shows particle size of NCM-VCR (A), DCM-VCR (B) and DCM-VCRafter cross-linking (C). TEM image of DCM-VCR after cross-linking (D)(scale bar: 50 nm). Vincristine (VCR) loading was 20:1 telodendrimer toVCR (w/w).

FIG. 16 shows particle size of NCM-VCR and DCM-VCR under micelledisrupting conditions. NCM-VCR (A) and DCM-VCR (C) were incubated in 50%human plasma (v/v) for 24 h at 37° C. Additionally, NCM-VCR (B) andDCM-VCR (D) were diluted to 2 mg/mL and incubated with 2.5 mg/mL SDS for30 min. DCM-VCR (E) was incubated with both SDS and 20 mMN-acetylcysteine (NAC).

FIG. 17 shows drug release profile of conventional VCR, NCM-VCR andDCM-VCR (A). VCR formulations were dialyzed against 1 L PBS at 37° C. inthe presence of 10 g/L charcoal to maintain sink conditions. The invitro cytotoxicity of conventional VCR, NCM-VCR and DCM-VCR was assessedin Raji cells treated for 72 h continuously (B) or 2 h, washed and thenincubated for 70 h (C). Cell viability was measured using an MTS assay.*, p<0.05; **, p<0.005.

FIG. 18 shows ex vivo near-infrared optical imaging of Raji tumorbearing mice intravenously injected with free DiD or DCM co-loaded withVCR and DiD (DCM-VCR/DiD). 72 h post injection, tumors and major organswere excised and imaged using an excitation/emission filter of 625/700nm.

FIG. 19 shows in vivo anti-tumor efficacy (A) and body weight loss (B)of Raji tumor bearing nude mice treated with PBS, conventional VCR (1mg/kg), DCM-VCR (1 mg/kg) plus or minus 100 mg/kg NAC or DCM-VCR (2.5mg/kg). Arrows indicate the days when mice were treated. *, p<0.05; **,p<0.005.

FIG. 20 shows mice (n=3) from the PBS (A), conventional VCR 1 mg/kg (B)and DCM-VCR 2.5 mg/kg (C) groups were sacrificed 8 days after the finaltreatment and the sciatic nerve was dissected. The nerves were processedinto epoxy blocks and 500 nm sections were cut, collected onto slidesand stained with Methylene Blue and Azur B stain.

FIG. 21 shows a schematic representation of the telodendrimer pair[PEG^(5k)-(boronic acid/catechol)₄-CA₈] and the resulting boronatecrosslinked micelles (BCM) in response to mannitol and/or acidic pH.

FIG. 22 shows (A) the fluorescent intensity of ARS (0.1 mM) upon mixingwith micelles formed by PEG^(5k)-NBA₄-CA₈(0.1 mM) with different ratiosof PEG^(5k)-Catechol₄-CA₈(0-0.5 mM) in PBS at pH7.4. Excitation: 468 nm.(B) Continuous dynamic light scattering measurements of NCM in SDS andBCM4 in SDS for 120 min, at which time mannitol was added or pH of thesolution was adjusted to 5.0 (see arrow). TEM images of BCM4 in PBS(C1),BCM4 in SDS for 120 min (C2), BCM4 in SDS for 120 mM and then adjustedthe pH of the solution to 5.0 for 20 min (C3), and BCM4 in SDS for 120min and then treated with mannitol (100 mM) for 20 min (C4), (scale bar:100 nm).

FIG. 23 shows (A) pH- and diol-responsive paclitaxel (PTX) releaseprofiles of BCM4 by treating with diols (mannitiol and glucose) and/orpH 5.0 at 5 hr compared with that of NCM. (B) MTT assays showing theviability of SKOV-3 cells after 1 hr incubation with Taxol®, PTX-NCM andPTX-BCM4 with or without treatment with 100 mM mannitol at pH5.0,followed by 3 times wash with PBS and additional 23 hr incubation. *:p<0.05, **: p<0.01, ***: p<0.001.

FIG. 24 shows schematic illustration of FRET-NCM in PBS (A) and in DMSO(B) at pH7.4; (C) Fluorescence emission spectra of FRET-NCM in PBS (redline) and DMSO (black line) with 480 nm excitation. (D) Emission spectraof FRET-BCM4 in PBS (red line) and DMSO (black line) with 480 nmexcitation. (E) The FRET ratio(I_(rhodamine B)/(I_(rhodamine B)+I_(DiO))) in blood of nude mice (n=3)over time after intravenous injection of 100 μl, FRET-NCM and FRET-BCM4(2.0 mg/mL). Excitation: 480 nm. (F) The fluorescence signal changes ofrhodamine B conjugated NCM and BCM4 in the blood collected at differenttime points after intravenous injection in the nude mice (n=3).Excitation: 540 nm. (G) Ex vivo near infrared fluorescence (NIRF) imagesof SKOV-3 xenograft bearing mouse obtained after intravenous injectionof BCM4 co-loaded with PTX and DiD.

FIG. 25 shows synthetic schemes (A, B and C) for the catechol containingtelodendrimers and boronic acid containing telodendrimers. The pinacolesters of boronic acid containing telodendrimers were removed viaDCM/TFA (1/1, v/v) at the last step. The synthetic scheme ofPEG^(5h)-BA2-CA₈ was shown as an example.

FIG. 26 shows the chemical structure of the catechol (A) containingtelodendrimers and boronic acid (B and C) containing telodendrimers.

FIG. 27 shows the MALDI-TOF MS of the starting PEG and therepresentative telodendrimer pair(PEG^(5k)-NBA₄-CA₈/PEG^(5k)-Catechol₄-CA₈) comparing with PEG^(5k)-CA₈telodendrimer. The pinacol ester form of PEG^(5k)-NBA₄-CA₈ was shown.

FIG. 28 shows ¹H NMR spectra of the representative telodendrimer pair(PEG^(5k)-NBA₄-CA₈/PEG^(5k)-Catechol₄-CA₈) comparing with thecorresponding small molecular 3-Carboxy-5-nitrophenylboronic acid and3,4-Dihydroxybenzoic acid as well as PEG^(5k)-CAS telodendrimer recordedin DMSO-d6.

FIG. 29 shows the particle size of NCM, BCM1, BCM2, BCM3 and BCM4,measured by dynamic light scattering (Microtrac®).

FIG. 30 shows the particle size of NCM in the absence (A) and in thepresence (B) of plasma 50% (v/v) for 24 h; The particle size of NCM inthe presence of 2.5 mg/mL SDS for 10 sec (C); The particle size of BCM4in the absence (D) and in the presence of plasma 50% (v/v) for 24 h (E);The particle size of BCM4 in the presence of 2.5 mg/mL SDS for 120 min(F); The particle size of BCM4 in SDS for 120 mM and then adjusted thepH of the solution to 5.0 for 20 min (G). The particle size of BCM4 inSDS for 120 min and then treated with mannitol (100 mM) for 20 min (H)The particle size of BCM4 in SDS for 120 min and then treated withglucose (100 mM) for 20 min (I). The particle size was measured bydynamic light scattering (Microtrac). The concentration of micelles waskept at 1.0 mg/mL in PBS.

FIG. 31 shows continuous particle size measurements of BCM4 in thepresence of 2.5 mg/mL SDS at pH 7.4 via DLS. The concentration ofmicelles was kept at 1.0 mg/mL.

FIG. 32 shows PTX release from NCM, BCM3 and BCM4 at 9 h (A) atdifferent pH levels and (B) in the presence of different concentrationsof mannitol (Man) or glucose (Glu). (C) Cumulative PTX release profilesof BCM4 at different pH levels (5.0 and 7.4) and in the presence ofmannitol (100 mM) compared with that of NCM at pH7.4.

FIG. 33 shows therapeutic efficacies (tumor size) of variousformulations of paclitaxel, non-cross-linked and boronate-catecholcross-inked nanoparticles, with and without mannitol given 24 hr aftereach dose of nanoparticle drug.

FIG. 34 shows intravenous dexamethasone (Dex) loaded into nanocarriersdecreases lung lavage eosinophil counts to a greater degree thanequivalent doses of iv Dex alone.

DETAILED DESCRIPTION OF THE INVENTION I. General

The present invention provides telodendrimers having crosslinking groupssuch that the nanocarrier micelles formed from the telodendrimers arecrosslinked to improve stability of the nanocarrier micelle. Thecrosslinking groups can be on the dendritic polymer itself, or on thelinking portion between the dendritic polymer and the PEG group. Anysuitable crosslinking group can be used, such as those capable ofreacting with themselves, or a complementary pair of functional groupsthat react with each other.

II. DEFINITIONS

As used herein, the terms “dendrimer” and “dendritic polymer” refer tobranched polymers containing a focal point, a plurality of branchedmonomer units, and a plurality of end groups. The monomers are linkedtogether to form arms (or “dendrons”) extending from the focal point andterminating at the end groups. The focal point of the dendrimer can beattached to other segments of the compounds of the invention, and theend groups may be further functionalized with additional chemicalmoieties.

As used herein, the term “telodendrimer” refers to a dendrimercontaining a hydrophilic PEG segment and one or more chemical moietiescovalently bonded to one or more end groups of the dendrimer. Thesemoieties can include, but are not limited to, hydrophobic groups,hydrophilic groups, amphiphilic compounds, and drugs. Different moietiesmay be selectively installed at a desired end group using orthogonalprotecting group strategies.

As used herein, the term “bow-tie dendrimer” or “bow-tie telodendrimer”refers to a dendrimer containing two branched segments, such as adendrimer and a branched PEG moiety, that are linked together at theirfocal points using a linker moiety.

As used herein, the term “nanocarrier” refers to a micelle resultingfrom aggregation of the dendrimer conjugates of the invention. Thenanocarrier has a hydrophobic core and a hydrophilic exterior.

As used herein, the terms “monomer” and “monomer unit” refer to adiamino carboxylic acid, a dihydroxy carboxylic acid and a hydroxylaminocarboxylic acid. Examples of diamino carboxylic acid groups of thepresent invention include, but are not limited to, 2,3-diamino propanoicacid, 2,4-diaminobutanoic acid, 2,5-diaminopentanoic acid (ornithine),2,6-diaminohexanoic acid (lysine), (2-Aminoethyl)-cysteine,3-amino-2-aminomethyl propanoic acid, 3-amino-2-aminomethyl-2-methylpropanoic acid, 4-amino-2-(2-aminoethyl)butyric acid and5-amino-2-(3-aminopropyl)pentanoic acid. Examples of dihydroxycarboxylic acid groups of the present invention include, but are notlimited to, glyceric acid, 2,4-dihydroxybutyric acid, glyceric acid,2,4-dihydroxybutyric acid, 2,2-Bis(hydroxymethyl)propionic acid and2,2-Bis(hydroxymethyl)butyric acid. Examples of hydroxylamino carboxylicacids include, but are not limited to, serine and homoserine. One ofskill in the art will appreciate that other monomer units are useful inthe present invention.

As used herein, the term “amino acid” refers to a carboxylic acidbearing an amine functional group. Amino acids include the diaminocarboxylic acids described above. Amino acids include naturallyoccurring α-amino acids, wherein the amine is bound to the carbonadjacent to the carbonyl carbon of the carboxylic acid. Examples ofnaturally occurring α-amino acids include, but are not limited to,L-aspartic acid, L-glutamic acid, L-histidine, L-lysine, and L-arginine.Amino acids may also include the D-enantiomers of naturally occurringα-amino acids, as well as β-amino acids and other non-naturallyoccurring amino acids.

As used herein, the term “linker” refers to a chemical moiety that linksone segment of a dendrimer conjugate to another. The types of bonds usedto link the linker to the segments of the dendrimers include, but arenot limited to, amides, amines, esters, carbamates, ureas, thioethers,thiocarbamates, thiocarbonate and thioureas. One of skill in the artwill appreciate that other types of bonds are useful in the presentinvention.

As used herein, the term “oligomer” refers to five or fewer monomers, asdescribed above, covalently linked together. The monomers may be linkedtogether in a linear or branched fashion. The oligomer may function as afocal point for a branched segment of a telodendrimer.

As used herein, the term “hydrophobic group” refers to a chemical moietythat is water-insoluble or repelled by water. Examples of hydrophobicgroups include, but are not limited to, long-chain alkanes and fattyacids, fluorocarbons, silicones, certain steroids such as cholesterol,and many polymers including, for example, polystyrene and polyisoprene.

As used herein, the term “hydrophilic group” refers to a chemical moietythat is water-soluble or attracted to water. Examples of hydrophilicgroups include, but are not limited to, alcohols, short-chain carboxylicacids, quaternary amines, sulfonates, phosphates, sugars, and certainpolymers such as PEG.

As used herein, the term “amphiphilic compound” refers to a compoundhaving both hydrophobic portions and hydrophilic portions. For example,the amphiphilic compounds of the present invention can have onehydrophilic face of the compound and one hydrophobic face of thecompound. Amphiphilic compounds useful in the present invention include,but are not limited to, cholic acid and cholic acid analogs andderivatives.

As used herein, the term “cholic acid” refers to(R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydrp-1H-cyclopenta[α]phenanthren-17-yl)pentanoicacid. Cholic acid is also known as 3α,7α,12α-trihydroxy-5β-cholanoicacid; 3-α,7-α,12-α-Trihydroxy-5-β-cholan-24-oic acid;17-β-(1-methyl-3-carboxypropyl)etiocholane-3α,7α,12α-triol; cholalicacid; and cholalin. Cholic acid derivatives and analogs, such asallocholic acid, pythocholic acid, avicholic acid, deoxycholic acid,chenodeoxycholic acid, are also useful in the present invention. Cholicacid derivatives can be designed to modulate the properties of thenanocarriers resulting from telodendrimer assembly, such as micellestability and membrane activity. For example, the cholic acidderivatives can have hydrophilic faces that are modified with one ormore glycerol groups, aminopropanediol groups, or other groups.

As used herein, the terms “drug” or “therapeutic agent” refers to anagent capable of treating and/or ameliorating a condition or disease. Adrug may be a hydrophobic drug, which is any drug that repels water.Hydrophobic drugs useful in the present invention include, but are notlimited to, paclitaxel, doxorubicin, etoposide, irinotecan, SN-38,cyclosporin A, podophyllotoxin, Carmustine, Amphotericin, Ixabepilone,Patupilone (epothelone class), rapamycin and platinum drugs. The drugsof the present invention also include prodrug forms. One of skill in theart will appreciate that other drugs are useful in the presentinvention.

As used herein, the term “crosslinkable group” or “crosslinking group”refers to a functional group capable of binding to a similar orcomplementary group on another molecule, for example, a firstcrosslinkable group on a first dendritic polymer linking to a secondcrosslinkable group on a second dendritic polymer. Groups suitable ascrosslinkable and crosslinking groups in the present invention includethiols such as cysteine, boronic acids and 1,2-diols including1,2-dihydroxybenzenes such as catechol. When the crosslinkable andcrosslinking groups combine, they form cross-linked bonds such asdisulfides and boronic esters. Other crosslinkable and crosslinkinggroups are suitable in the present invention.

As used herein, the term “bond cleavage component” refers to an agentcapable of cleaving the cross-linked bonds formed using thecrosslinkable and crosslinking groups of the present invention. The bondcleavage component can be a reducing agent, such as glutathione, whenthe cross-linked bond is a disulfide, or mannitol when the cross-linkedbond is formed from a boronic acid and 1,2-diol.

As used herein, the term “imaging agent” refers to chemicals that allowbody organs, tissue or systems to be imaged. Exemplary imaging agentsinclude paramagnetic agents, optical probes, and radionuclides.

As used herein, the terms “treat”, “treating” and “treatment” refers toany indicia of success in the treatment or amelioration of an injury,pathology, condition, or symptom (e.g., pain), including any objectiveor subjective parameter such as abatement; remission; diminishing ofsymptoms or making the symptom, injury, pathology or condition moretolerable to the patient; decreasing the frequency or duration of thesymptom or condition; or, in some situations, preventing the onset ofthe symptom or condition. The treatment or amelioration of symptoms canbe based on any objective or subjective parameter; including, e.g., theresult of a physical examination.

As used herein, the term “subject” refers to animals such as mammals,including, but not limited to, primates (e.g., humans), cows, sheep,goats, horses, dogs, cats, rabbits, rats, mice and the like. In certainembodiments, the subject is a human.

As used herein, the terms “therapeutically effective amount or dose” or“therapeutically sufficient amount or dose” or “effective or sufficientamount or dose” refer to a dose that produces therapeutic effects forwhich it is administered. The exact dose will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003,Gennaro, Ed., Lippincott, Williams & Wilkins). In sensitized cells, thetherapeutically effective dose can often be lower than the conventionaltherapeutically effective dose for non-sensitized cells.

III. TELODENDRIMERS

The present invention provides crosslinkable telodendrimer conjugateshaving a hydrophilic poly(ethylene glycol) (PEG) segment and ahydrophobic segment. The PEG segment can have a branched or lineararchitecture including one or more PEG chains. The hydrophobic segmentof the telodendrimer can be provided by cholic acid, which has ahydrophobic face and a hydrophilic face. The cholic acid and the PEG areconnected by oligomers and/or polymers that can contain a variety ofacid repeats units. Typically, the oligomers and polymers comprise adiamino carboxylic acid, lysine. The telodendrimers are alsofunctionalized with a crosslinkable group. The telodendrimers canaggregate in solution to form micelles with a hydrophobic interior and ahydrophilic exterior, and can be used as nanocarriers to deliver drugsor other agents having low water solubility. Following micelleformation, the telodendrimers can be crosslinked using the crosslinkablegroups, forming a more stable micelle.

The present invention provides a PEGylated dendrimer, referred to as atelodendrimer, containing cholic acid groups and other moieties at thedendrimer periphery, and crosslinkable groups. In some embodiments, theinvention provide a compound of formula I:

(PEG)_(m)-A(Y¹)_(p)-L-D(Y²)_(q)—(R)_(n)  (I)

wherein radical A of formula I is linked to at least one PEG group.Radical D of formula I is a dendritic polymer having a single focalpoint group, a plurality of branched monomer units X and a plurality ofend groups. Radical L of formula I is a bond or a linker linked to thefocal point group of the dendritic polymer. Each PEG of formula I is apolyethyleneglycol (PEG) polymer, wherein each PEG polymer has amolecular weight of 1-100 kDa. Each R of formula I can be the end groupof the dendritic polymer, a hydrophobic group, a hydrophilic group, anamphiphilic compound or a drug, such that when R is not an end groupthen each R is linked to one of the end groups. Each Y¹ and Y² offormula I is a crosslinkable group that can be any of boronic acid,dihydroxybenzene or a thiol. Subscript m of formula I is an integer from0 to 20. Subscript n of formula I is an integer from 2 to 20, whereinsubscript n is equal to the number of end groups on the dendriticpolymer, and wherein at least half the number n of R groups that caneach be a hydrophobic group, a hydrophilic group, an amphiphiliccompound or a drug. And, each of subscripts p and q are 0 or from 2 to8, such that one of subscripts p and q is from 2 to 8.

Radical A can be any suitable group capable of linking the PEG to thelinker or dendritic polymer D. Suitable A groups include the monomerunits X described below for the dendritic polymer. In some embodiments,radical A is a monomer or oligomer of lysine. In some embodiments,radical A is lysine. Radical A can be linked to a crosslinkable group oran imaging agent, either directly or via a linker. In some embodiments,radical A can also include at least one imaging agent. Imaging agentsuseful for attachment to the telodendrimers of the present inventioninclude, but are not limited to, fluorescent dyes, chelates andradiometals.

The dendritic polymer can be any suitable dendritic polymer. Thedendritic polymer can be made of branched monomer units including aminoacids or other bifunctional AB2-type monomers, where A and B are twodifferent functional groups capable of reacting together such that theresulting polymer chain has a branch point where an A—B bond is formed.In some embodiments, each branched monomer unit X can be a diaminocarboxylic acid, a dihydroxy carboxylic acid and a hydroxylaminocarboxylic acid. In some embodiments, each diamino carboxylic acid canbe 2,3-diamino propanoic acid, 2,4-diaminobutanoic acid,2,5-diaminopentanoic acid (ornithine), 2,6-diaminohexanoic acid(lysine), (2-Aminoethyl)-cysteine, 3-amino-2-aminomethyl propanoic acid,3-amino-2-aminomethyl-2-methyl propanoic acid,4-amino-2-(2-aminoethyl)butyric acid or5-amino-2-(3-aminopropyl)pentanoic acid. In some embodiments, eachdihydroxy carboxylic acid can be glyceric acid, 2,4-dihydroxybutyricacid, 2,2-Bis(hydroxymethyl)propionic acid,2,2-Bis(hydroxymethyl)butyric acid, serine or threonine. In someembodiments, each hydroxyl amino carboxylic acid can be serine orhomoserine. In some embodiments, the diamino carboxylic acid is an aminoacid. In some embodiments, each branched monomer unit X is lysine.

The dendritic polymer of the telodendrimer can be any suitablegeneration of dendrimer, including generation 1, 2, 3, 4, 5, or more,where each “generation” of dendrimer refers to the number of branchpoints encountered between the focal point and the end group followingone branch of the dendrimer. The dendritic polymer of the telodendrimercan also include partial-generations such as 1.5, 2.5, 3.5, 4.5, 5.5,etc., where a branch point of the dendrimer has only a single branch.The various architectures of the dendritic polymer can provide anysuitable number of end groups, including, but not limited to, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31 or 32 end groups.

The focal point of a telodendrimer or a telodendrimer segment may be anysuitable functional group. In some embodiments, the focal point includesa functional group that allows for attachment of the telodendrimer ortelodendrimer segment to another segment. The focal point functionalgroup can be a nucleophilic group including, but not limited to, analcohol, an amine, a thiol, or a hydrazine. The focal point functionalgroup may also be an electrophile such as an aldehyde, a carboxylicacid, or a carboxylic acid derivative including an acid chloride or anN-hydroxysuccinimidyl ester.

The R groups installed at the telodendrimer periphery can be anysuitable chemical moiety, including hydrophilic groups, hydrophobicgroups, or amphiphilic compounds. Examples of hydrophobic groupsinclude, but are not limited to, long-chain alkanes and fatty acids,fluorocarbons, silicones, certain steroids such as cholesterol, and manypolymers including, for example, polystyrene and polyisoprene. Examplesof hydrophilic groups include, but are not limited to, alcohols,short-chain carboxylic acids, amines, sulfonates, phosphates, sugars,and certain polymers such as PEG. Examples of amphiphilic compoundsinclude, but are not limited to, molecules that have one hydrophilicface and one hydrophobic face.

Amphiphilic compounds useful in the present invention include, but arenot limited to, cholic acid and cholic acid analogs and derivatives.“Cholic acid” refers to(R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[α]phenanthren-17-yl)pentanoicacid, having the structure:

Cholic acid derivatives and analogs include, but are not limited to,allocholic acid, pythocholic acid, avicholic acid, deoxycholic acid, andchenodeoxycholic acid. Cholic acid derivatives can be designed tomodulate the properties of the nanocarriers resulting from telodendrimerassembly, such as micelle stability and membrane activity. For example,the cholic acid derivatives can have hydrophilic faces that are modifiedwith one or more glycerol groups, aminopropanediol groups, or othergroups.

Telodendrimer end groups may also include drugs such as paclitaxel,doxorubicin, etoposide, irinotecan, SN-38, cyclosporin A,podophyllotoxin, carmustine, amphotericin, ixabepilone, patupilone(epothelone class), rapamycin and platinum drugs. One of skill in theart will appreciate that other drugs are useful in the presentinvention.

In some embodiments, each R can be cholic acid, (3α, 5β,7α,12α)-7,12-dihydroxy-3-(2,3-dihydroxy-1-propoxy)-cholic acid, (3α,5β,7α,12α)-7-hydroxy-3,12-di(2,3-dihydroxy-1-propoxy)-cholic acid, (3α,5β,7α,12α)-7,12-dihydroxy-3-(3-amino-2-hydroxy-1-propoxy)-cholic acid,cholesterol formate, doxorubicin, or rhein. In some embodiments, each Rcan be cholic acid.

The telodendrimer backbone can vary, depending on the number of branchesand the number and chemical nature of the end groups and R groups, whichwill modulate solution conformation, rheological properties, and othercharacteristics. The telodendrimers can have any suitable number n ofend groups and any suitable number of R groups. In some embodiments, ncan be 2-70, or 2-50, or 2-30, or 2-10. Subscript n can be about 2, 3,4, 5, 6, 7, 8, 9, 10, 15, or 20. In some embodiments, subscript n can befrom 2 to 10, 2 to 8, or 4 to 8. In some embodiment, n is 2-20.

The telodendrimer can have a single type of R group on the periphery, orany combination of R groups in any suitable ratio. For example, at leasthalf the number n of R groups can be a hydrophobic group, a hydrophilicgroup, an amphiphilic compound, a drug, or any combination thereof. Insome embodiments, half the number n of R groups are amphiphiliccompounds.

In some embodiments, each R group is the same. In some embodiments, atleast two different R groups are present, such as two differentamphiphilic groups, or an amphiphilic group and a drug, or anamphiphilic group and a dendritic polymer end group, or two differentdrugs, or a drug and a dendritic end group.

The linker L can include any suitable linker. In general, the linkersare bifunctional linkers, having two functional groups for reaction witheach of two telodendrimer segments. In some embodiments, the linker canbe a heterobifunctional linker. In some embodiments, the linker can be ahomobifunctional linker. In some embodiments, the linker L can bepolyethylene glycol, polyserine, polyglycine, poly(serine-glycine),aliphatic amino acids, 6-amino hexanoic acid, 5-amino pentanoic acid,4-amino butanoic acid or beta-alanine. One of skill in the art willrecognize that the size and chemical nature of the linker can be variedbased on the structures of the telodendrimer segments to be linked.

In some embodiments, linker L is the Ebes linker having the formula:

Polyethylene glycol (PEG) polymers of any size and architecture areuseful in the nanocarriers of the present invention. In someembodiments, the PEG is from 1-100 kDa. In other embodiments, the PEG isfrom 1-10 kDa. In some other embodiments, the PEG is about 3 kDa. Instill other embodiments, additional PEG polymers are linked to theamphiphilic compounds. For example, when the amphiphilic compound ischolic acid, up to 3 PEG polymers are linked to each cholic acid. ThePEG polymers linked to the amphiphilic compounds are from 200-10,000 Dain size. In yet other embodiments, the PEG polymers linked to theamphiphilic compounds are from 1-5 kDa in size. One of skill in the artwill appreciate that other PEG polymers and other hydrophilic polymersare useful in the present invention. PEG can be any suitable length.

Any suitable number of PEG groups can be present. For example, subscriptm can be 0, 1, 2, 3, 4, 5, 10, 15, or 20. Subscript m can also be from 0to 5, 0 to 4, 0 to 3, 0 to 2, 1 to 5, 1 to 4, or 1 to 3. In someembodiments, subscript m is 1.

Crosslinkable groups suitable in the compounds of the present inventioninclude any functional group capable of forming a covalent bond with thesame functional group on another telodendrimer, or with a complementaryfunctional group on another telodendrimer. Functional groups capable offorming a covalent bond with the same functional group include thiols.Thiols useful in the compounds of the present invention include anythiols, such as cysteine.

Complementary functional groups capable of forming a covalent bondinclude boronic acid and a 1,2-diol. Boronic acids useful in thecompounds of the present invention include, but are not limited to,phenylboronic acid, 2-thienylboronic acid, methylboronic acid, andpropenylboronic acid. Suitable 1,2-diols include alkyl-1,2-diol andphenyl-1,2-diols such as catechol.

In some embodiments, each crosslinkable group Y¹ and Y² can be any ofboronic acid, dihydroxybenzene or a thiol. In some embodiments, eachcrosslinkable group Y¹ and Y² can be any of boronic acid ordihydroxybenzene. In some embodiments, each crosslinkable group Y¹ andY² can be phenylboronic acid or dihydroxybenzene. In some embodiments,each crosslinkable group Y′ can be phenylboronic acid ordihydroxybenzene. In some embodiments, each crosslinkable group Y′ canbe carboxyphenylboronic acid, carboxynitrophenyl boronic acid or3,4-dihydroxybenzoic acid. In some embodiments, each crosslinkable groupY¹ and Y² can be a thiol. In some embodiments, each crosslinkable groupY¹ and Y² can be cysteine. In some embodiments, each crosslinkable groupY² can be cysteine.

In some embodiments, the compound of formula I has the structure:

PEG-A(Y¹)_(p)-D-(R)_(n)  (Ia)

wherein subscript p is an integer from 2 to 8 and subscript q is 0.

In some embodiments, the compound of formula has the structure:

wherein each L′ is a linker Ebes, PEG is PEGSk, each R is cholic acid,each branched monomer unit X is lysine, and Y¹ can becarboxyphenylboronic acid, carboxynitrophenyl boronic acid and3,4-dihydroxybenzoic acid.

In other embodiments, the compound of formula I has the structure:

PEG-A-D(Y²)_(q)—(R)_(n)  (Ib)

wherein subscript p is 0 and subscript q is an integer from 2 to 8.

In some embodiments, the compound of formula has the structure:

wherein A is lysine, each L′ is a linker Ebes, PEG is PEG5k, each R ischolic acid, each branched monomer unit X is lysine, and each Y² iscysteine.

The compounds and conjugates of the present invention can be prepared bymethods known to one of skill in the art. For example, thewell-established stepwise Fmoc peptide chemistry method was employed inthe preparation of the compounds and conjugates of the presentinvention, with the resulting thiolated telodendrimers havingwell-defined polymer structure. In one example, the compound andconjugate is designated as PEG^(5k)-Cys₄-L₈-CA₈ corresponding to lengthof PEG and the number of cysteines, hydrophilic spacers and cholic acidsin the structure. As shown in the figures, PEG^(5k)-Cys₄-L₈-CA₈ includesa dendritic oligomer of cholic acids attached to one terminus of thelinear PEG through a poly(lysine-cysteine-Ebes) backbone. The thiol freetelodendrimer, PEG^(5k)-CA₈ was also synthesized for comparison asdescribed previously. Fluorescent dyes such as BODIPY can be attached tothe E-amino group of the lysine at the junction between the PEG and theoligo-cholic acid chains after removal of Dde protecting group.

IV. NANOCARRIERS

The telodendrimers of the present invention aggregate to formnanocarriers with a hydrophobic core and a hydrophilic exterior, wherethe crosslinkable groups are subsequently crosslinked to provideadditional stability to the resulting nanocarrier.

In some embodiments, the invention provides a nanocarrier having aninterior and an exterior, the nanocarrier including at least twoconjugates, wherein each conjugate includes a polyethylene glycol (PEG)polymer, at least two amphiphilic compounds having both a hydrophilicface and a hydrophobic face, at least two crosslinking groups, and adendritic polymer covalently attached to the PEG, the amphiphiliccompounds and the crosslinking groups, wherein each conjugateself-assembles in an aqueous solvent to form the nanocarrier such that ahydrophobic pocket is formed in the interior of the nanocarrier, whereinthe PEG of each compound self-assembles on the exterior of thenanocarrier, and wherein at least two conjugates are reversiblycrosslinked via the crosslinking groups.

In some embodiments, each conjugate can be a conjugate of the presentinvention.

The crosslinking groups can be any suitable crosslinking group, asdescribed above. In some embodiments, the crosslinking groups can bethiol, boronic acid or dihydroxybenzene. In some embodiments, thecrosslinking groups can be thiol. In some embodiments, a first set ofconjugates includes boronic acid crosslinking groups, and a second setof conjugates includes dihydroxybenzene crosslinking groups.

In some embodiments, the nanocarrier also includes a hydrophobic drug oran imaging agent, such that the hydrophobic drug or imaging agent issequestered in the hydrophobic pocket of the nanocarrier. Hydrophobicdrugs useful in the nanocarrier of the present invention includes anydrug having low water solubility. In some embodiments, the hydrophobicdrug in the nanocarrier can be bortezomib, paclitaxel, SN38,camptothecin, etoposide and doxorubicin, docetaxel, daunorubicin, VP16,prednisone, dexamethasone, vincristine, vinblastine, temsirolimus andcarmusine.

In some embodiments, the nanocarrier includes at least one monomer unitthat is optionally linked to an optical probe, a radionuclide, aparamagnetic agent, a metal chelate or a drug. The drug can be a varietyof hydrophilic or hydrophobic drugs, and is not limited to thehydrophobic drugs that are sequestered in the interior of thenanocarriers of the present invention.

Drugs that can be sequestered in the nanocarriers or linked to theconjugates of the present invention include, but are not limited to,cytostatic agents, cytotoxic agents (such as for example, but notlimited to, DNA interactive agents (such as cisplatin or doxorubicin));taxanes (e.g. taxotere, taxol); topoisomerase II inhibitors (such asetoposide); topoisomerase I inhibitors (such as irinotecan (or CPT-11),cam ptostar, or topotecan); tubulin interacting agents (such aspaclitaxel, docetaxel or the epothilones); hormonal agents (such astamoxifen); thymidilate synthase inhibitors (such as 5-fluorouracil);anti-metabolites (such as methotrexate); alkylating agents (such astemozolomide (TEMODAR™ from Schering-Plough Corporation, Kenilworth,N.J.), cyclophosphamide); aromatase combinations; ara-C, adriamycin,cytoxan, and gemcitabine. Other drugs useful in the nanocarrier of thepresent invention include but are not limited to Uracil mustard,Chlormethine, Ifosfamide, Melphalan, Chlorambucil, Pipobroman,Triethylenemelamine, Triethylenethiophosphoramine, Busulfan, Carmustine,Lomustine, Streptozocin, Dacarbazine, Floxuridine, Cytarabine,6-Mercaptopurine, 6-Thioguanine, Fludarabine phosphate, oxaliplatin,leucovirin, oxaliplatin (ELOXATIN™ from Sanofi-SynthelaboPharmaceuticals, France), Pentostatine, Vinblastine, Vincristine,Vindesine, Bleomycin, Dactinomycin, Daunorubicin, Doxorubicin,Epirubicin, Idarubicin, Mithramycin, Deoxycoformycin, Mitomycin-C,L-Asparaginase, Teniposide 17.alpha.-Ethinylestradiol,Diethylstilbestrol, Testosterone, Prednisone, Fluoxymesterone,Dromostanolone propionate, Testolactone, Megestrolacetate,Methylprednisolone, Methyltestosterone, Prednisolone, Triamcinolone,Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide, Estramustine,Medroxyprogesteroneacetate, Leuprolide, Flutamide, Toremifene,goserelin, Cisplatin, Carboplatin, Hydroxyurea, Amsacrine, Procarbazine,Mitotane, Mitoxantrone, Levamisole, Navelbene, Anastrazole, Letrazole,Capecitabine, Reloxafine, Droloxafine, or Hexamethylmelamine. Prodrugforms are also useful in the present invention.

Other drugs useful in the present invention also include radionuclides,such as ⁶⁷Cu, ⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁸⁸Re, ¹⁸⁶Re and ²¹¹At. Insome embodiments, a radionuclide can act therapeutically as a drug andas an imaging agent.

Imaging agents include paramagnetic agents, optical probes andradionuclides. Paramagnetic agents include iron particles, such as ironnanoparticles that are sequestered in the hydrophobic pocket of thenanocarrier. In some embodiments, the imaging agent can include organicfluorescent dyes, quantum dots (QDs), super paramagnetic iron oxidenanoparticles (SPIO-NPs), or gold nanoparticles. In some embodiments,the imaging agent is a radionuclide.

Some embodiments of the invention provide nanocarriers wherein eachamphiphilic compound can be any of cholic acid, allocholic acid,pythocholic acid, avicholic acid, deoxycholic acid, or chenodeoxycholicacid. In some embodiments, each amphiphilic compound can be cholic acid.

The crosslinking groups of the nanocarrier telodendrimers can becrosslinked by any suitable means known to one of skill in the art. Forexample, when the crosslinkable group is a thiol, a disulfide bond canbe formed between two conjugates of the present invention by oxidationof the thiols. Any suitable oxidation agent can be used, such as oxygen.For other crosslinking groups, such as the complementary groups boronicacid and 1,2-diols such as dihydroxybenzene, the crosslinking occursspontaneously and without the need for additional reactants.

The crosslinking in the nanocarriers of the present invention isreversible to facilitate delivery of a drug, for example, to a targetsite. Reversing the crosslinking of the crosslinked nanocarrier requirescontacting the crosslinked nanocarrier with a suitable bond cleavagecomponent. In some embodiments, the present invention provides a methodof reversing the cross-linking of the reversibly crosslinked nanocarrierof the present invention, by contacting the reversibly crosslinkednanocarrier with a bond cleavage component suitable for cleaving thecross-linked bond, thereby reversing the cross-linking of the reversiblycrosslinked nanocarrier. In some embodiments, the contacting isperformed in vivo.

Any suitable bond cleavage component can be used in the presentinvention. In some embodiments, the bond cleavage component can beN-acetyl cysteine (NAC), glutathione, 2-mercaptoethane sulfonate sodium(MESNA), mannitol or acid. When the crosslinked bond is a disulfide, anydisulfide reducing agent is suitable. In some embodiments, the bondcleavage component can be N-acetyl cysteine or 2-mercaptoethanesulfonate when the compounds and conjugates of the present inventioninclude a thiol. In some embodiments, the bond cleavage component can bemannitol when the crosslinking groups of the compounds and conjugates ofthe present invention include boronic acid and dihydroxybenzene.

V. METHOD OF TREATING

The nanocarriers of the present invention can be used to treat anydisease requiring the administration of a drug, such as by sequesteringa hydrophobic drug in the interior of the nanocarrier, or by covalentattachment of a drug to a conjugate of the nanocarrier. The nanocarrierscan also be used for imaging, by sequestering an imaging agent in theinterior of the nanocarrier, or by attaching the imaging agent to aconjugate of the nanocarrier.

In some embodiments, the present invention provides a method of treatinga disease, including administering to a subject in need of suchtreatment, a therapeutically effective amount of a nanocarrier of thepresent invention, wherein the nanocarrier includes a drug. The drug canbe a covalently attached to a conjugate of the nanocarrier. In someembodiments, the drug is a hydrophobic drug sequestered in the interiorof the nanocarrier.

Any suitable drug can be used with the nanocarriers of the presentinvention. In some embodiments, the hydrophobic drug can be bortezomib,paclitaxel, SN38, camptothecin, etoposide and doxorubicin, docetaxel,daunorubicin, VP16, prednisone, dexamethasone, vincristine, vinblastine,temsirolimus, carmusine, lapatinib, sorafenib, fenretinide, oractinomycin D.

In some embodiments, the nanocarrier also includes an imaging agent. Theimaging agent can be a covalently attached to a conjugate of thenanocarrier, or the imaging agent can be sequestered in the interior ofthe nanocarrier. In some other embodiments, both a hydrophobic drug andan imaging agent are sequestered in the interior of the nanocarrier. Instill other embodiments, both a drug and an imaging agent are covalentlylinked to a conjugate or conjugates of the nanocarrier. In yet otherembodiments, the nanocarrier can also include a radionuclide.

The nanocarriers of the present invention can be administered to asubject for treatment, e.g., of hyperproliferative disorders includingcancer such as, but not limited to: carcinomas, gliomas, mesotheliomas,melanomas, lymphomas, leukemias, adenocarcinomas, breast cancer, ovariancancer, cervical cancer, glioblastoma, leukemia, lymphoma, prostatecancer, and Burkitt's lymphoma, head and neck cancer, colon cancer,colorectal cancer, non-small cell lung cancer, small cell lung cancer,cancer of the esophagus, stomach cancer, pancreatic cancer,hepatobiliary cancer, cancer of the gallbladder, cancer of the smallintestine, rectal cancer, kidney cancer, bladder cancer, prostatecancer, penile cancer, urethral cancer, testicular cancer, cervicalcancer, vaginal cancer, uterine cancer, ovarian cancer, thyroid cancer,parathyroid cancer, adrenal cancer, pancreatic endocrine cancer,carcinoid cancer, bone cancer, skin cancer, retinoblastomas, multiplemyelomas, Hodgkin's lymphoma, and non-Hodgkin's lymphoma (see, CANCER:PRINCIPLES AND PRACTICE (DeVita, V. T. et al. eds 2008) for additionalcancers). In some embodiments, the disease is cancer.

Other diseases that can be treated by the nanocarriers of the presentinvention include: (1) inflammatory or allergic diseases such assystemic anaphylaxis or hypersensitivity responses, drug allergies,insect sting allergies; inflammatory bowel diseases, such as Crohn'sdisease, ulcerative colitis, ileitis and enteritis; vaginitis; psoriasisand inflammatory dermatoses such as dermatitis, eczema, atopicdermatitis, allergic contact dermatitis, urticaria; vasculitis;spondyloarthropathies; scleroderma; respiratory allergic diseases suchas asthma, allergic rhinitis, hypersensitivity lung diseases, and thelike, (2) autoimmune diseases, such as arthritis (rheumatoid andpsoriatic), osteoarthritis, multiple sclerosis, systemic lupuserythematosus, diabetes mellitus, glomerulonephritis, and the like, (3)graft rejection (including allograft rejection and graft-v-hostdisease), and (4) other diseases in which undesired inflammatoryresponses are to be inhibited (e.g., atherosclerosis, myositis,neurological conditions such as stroke and closed-head injuries,neurodegenerative diseases, Alzheimer's disease, encephalitis,meningitis, osteoporosis, gout, hepatitis, nephritis, sepsis,sarcoidosis, conjunctivitis, otitis, chronic obstructive pulmonarydisease, sinusitis and Behcet's syndrome). In some embodiments, thedisease is asthma.

In addition, the nanocarriers of the present invention are useful forthe treatment of infection by pathogens such as viruses, bacteria,fungi, and parasites. Other diseases can be treated using thenanocarriers of the present invention.

A. Formulations

The nanocarriers of the present invention can be formulated in a varietyof different manners known to one of skill in the art. Pharmaceuticallyacceptable carriers are determined in part by the particular compositionbeing administered, as well as by the particular method used toadminister the composition. Accordingly, there are a wide variety ofsuitable formulations of pharmaceutical compositions of the presentinvention (see, e.g., Remington's Pharmaceutical Sciences, 20^(th) ed.,2003, supra). Effective formulations include oral and nasalformulations, formulations for parenteral administration, andcompositions formulated for with extended release.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of a compound of the presentinvention suspended in diluents, such as water, saline or PEG 400; (b)capsules, sachets, depots or tablets, each containing a predeterminedamount of the active ingredient, as liquids, solids, granules orgelatin; (c) suspensions in an appropriate liquid; (d) suitableemulsions; and (e) patches. The liquid solutions described above can besterile solutions. The pharmaceutical forms can include one or more oflactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch,potato starch, microcrystalline cellulose, gelatin, colloidal silicondioxide, talc, magnesium stearate, stearic acid, and other excipients,colorants, fillers, binders, diluents, buffering agents, moisteningagents, preservatives, flavoring agents, dyes, disintegrating agents,and pharmaceutically compatible carriers. Lozenge forms can comprise theactive ingredient in a flavor, e.g., sucrose, as well as pastillescomprising the active ingredient in an inert base, such as gelatin andglycerin or sucrose and acacia emulsions, gels, and the like containing,in addition to the active ingredient, carriers known in the art.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form. The composition can, if desired, also contain othercompatible therapeutic agents. Preferred pharmaceutical preparations candeliver the compounds of the invention in a sustained releaseformulation.

Pharmaceutical preparations useful in the present invention also includeextended-release formulations. In some embodiments, extended-releaseformulations useful in the present invention are described in U.S. Pat.No. 6,699,508, which can be prepared according to U.S. Pat. No.7,125,567, both patents incorporated herein by reference.

The pharmaceutical preparations are typically delivered to a mammal,including humans and non-human mammals. Non-human mammals treated usingthe present methods include domesticated animals (i.e., canine, feline,murine, rodentia, and lagomorpha) and agricultural animals (bovine,equine, ovine, porcine).

In practicing the methods of the present invention, the pharmaceuticalcompositions can be used alone, or in combination with other therapeuticor diagnostic agents.

B. Administration

The nanocarriers of the present invention can be administered asfrequently as necessary, including hourly, daily, weekly or monthly. Thecompounds utilized in the pharmaceutical method of the invention areadministered at the initial dosage of about 0.0001 mg/kg to about 1000mg/kg daily. A daily dose range of about 0.01 mg/kg to about 500 mg/kg,or about 0.1 mg/kg to about 200 mg/kg, or about 1 mg/kg to about 100mg/kg, or about 10 mg/kg to about 50 mg/kg, can be used. The dosages,however, may be varied depending upon the requirements of the patient,the severity of the condition being treated, and the compound beingemployed. For example, dosages can be empirically determined consideringthe type and stage of disease diagnosed in a particular patient. Thedose administered to a patient, in the context of the present inventionshould be sufficient to effect a beneficial therapeutic response in thepatient over time. The size of the dose also will be determined by theexistence, nature, and extent of any adverse side-effects that accompanythe administration of a particular compound in a particular patient.Determination of the proper dosage for a particular situation is withinthe skill of the practitioner. Generally, treatment is initiated withsmaller dosages which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small increments until theoptimum effect under circumstances is reached. For convenience, thetotal daily dosage may be divided and administered in portions duringthe day, if desired. Doses can be given daily, or on alternate days, asdetermined by the treating physician. Doses can also be given on aregular or continuous basis over longer periods of time (weeks, monthsor years), such as through the use of a subdermal capsule, sachet ordepot, or via a patch or pump.

The pharmaceutical compositions can be administered to the patient in avariety of ways, including topically, parenterally, intravenously,intradermally, subcutaneously, intramuscularly, colonically, rectally orintraperitoneally. Preferably, the pharmaceutical compositions areadministered parenterally, topically, intravenously, intramuscularly,subcutaneously, orally, or nasally, such as via inhalation.

In practicing the methods of the present invention, the pharmaceuticalcompositions can be used alone, or in combination with other therapeuticor diagnostic agents. The additional drugs used in the combinationprotocols of the present invention can be administered separately or oneor more of the drugs used in the combination protocols can beadministered together, such as in an admixture. Where one or more drugsare administered separately, the timing and schedule of administrationof each drug can vary. The other therapeutic or diagnostic agents can beadministered at the same time as the compounds of the present invention,separately or at different times.

The nanocarriers of the present invention can be used to administer anysuitable drug to a subject in need of treatment. In some embodiments,the present invention provides a method of delivering a drug to asubject in need thereof by administering a nanocarrier of the presentinvention to the subject, wherein the nanocarrier includes the drug anda plurality of cross-linked bonds. The method also includes cleaving thecross-linked bonds using a bond cleavage component, such that the drugis released from the nanocarrier, thereby delivering the drug to thesubject. Any suitable bond cleavage component can be used, as describedabove.

VI. METHOD OF IMAGING

In some embodiments, the present invention provides a method of imaging,including administering to a subject to be imaged, an effective amountof a nanocarrier of the present invention, wherein the nanocarrierincludes an imaging agent. In other embodiments, the method of treatingand the method of imaging are accomplished simultaneously using ananocarrier having both a drug and an imaging agent.

Exemplary imaging agents include paramagnetic agents, optical probes,and radionuclides. Paramagnetic agents imaging agents that are magneticunder an externally applied field. Examples of paramagnetic agentsinclude, but are not limited to, iron particles including nanoparticles.Optical probes are fluorescent compounds that can be detected byexcitation at one wavelength of radiation and detection at a second,different, wavelength of radiation. Optical probes useful in the presentinvention include, but are not limited to, Cy5.5, Alexa 680, Cy5, DiD(1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate)and DiR (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanineiodide). Other optical probes include quantum dots. Radionuclides areelements that undergo radioactive decay. Radionuclides useful in thepresent invention include, but are not limited to, ³H, ¹¹C, ¹³N, ¹⁸F,¹⁹F, ⁶⁰Co, ⁶⁴Cu, ⁶⁷Cu, ⁶⁸Ga, ⁸²Rb, ⁹⁰Sr, ⁹⁰Y, ⁹⁰Tc, ^(99m)Tc, ¹¹¹In,¹²³I, ¹²⁴I, ¹²⁵I, ¹²⁹I, ¹³¹I, ¹³⁷Cs, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, Rn, Ra,Th, U, Pu and ²⁴¹ Am.

VII. EXAMPLES Materials

Monomethylterminated poly(ethylene glycol)monoamine (MeO-PEG-NH₂, M_(w):5000 Da) was purchased from Rapp Polymere (Germany). PTX was purchasedfrom AK Scientific Inc. (Mountain View, Calif.). Taxol (Mayne Pharma,Paramus, N.J.) was obtained from the Cancer Center of University ofCalifornia, Davis. Vincristine sulfate was purchased from AvaChemScientific (San Antonio, Tex.). The conventional (clinical) formulationof vincristine sulfate was obtained from the Cancer Center of Universityof California, Davis. (Fmoc)lys(Boc)-OH, (Fmoc)Lys(Dde)-OH,(Fmoc)Lys(Fmoc)-OH, (Fmoc)Cys(Trt)-OH and (Fmoc)Ebes-OH were obtainedfrom AnaSpec Inc. (San Jose, Calif.).1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate(DiD), BODIPY650/665 and 4,6-diamidino-2-phenylindole (DAPI, blue) werepurchased from Invitrogen. Tetrazolium compound[3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, MTS] andphenazine methosulfate (PMS) were purchased from Promega (Madison,Wis.). Cholic acid, MTT [3-(4,5-dimethyldiazol-2-yl)-2,5diphenyltetrazolium bromide], Ellman's reagent [DTNB,5,59-dithiobis(2-nitrobenzoic acid)] and all other chemicals werepurchased from Sigma-Aldrich (St. Louis). Non-crosslinked micelle (NCM),disulfide crosslinked micelle (DCM) and boronate crosslinked micelle(BCM).

Animal Model

Female athymic nude mice (Nu/Nu strain), 6-8 weeks age, were purchasedfrom Harlan (Livermore, Calif.). All animals were kept underpathogen-free conditions according to AAALAC guidelines and were allowedto acclimatize for at least 4 days prior to any experiments. All animalexperiments were performed in compliance with institutional guidelinesand according to protocol No. 07-13119 and No. 09-15584 approved by theAnimal Use and Care

Statistical Analysis

Statistical analysis was performed by Student's t-test for two groups,and one-way ANOVA for multiple groups. All results were expressed as themean±standard error (SEM) unless otherwise noted. A value of P<0.05 wasconsidered statistically significant.

Example 1 Preparation of Thiolated Conjugate (PEG^(5k)-Cys₄-L₈-CA₈)

The thiolated telodendrimer (named as PEG^(5k)-Cys₄-L₈-CA₈) wassynthesized via solution-phase condensation reactions from MeO-PEG-NH₂utilizing stepwise peptide chemistry. The typical procedure forsynthesis of PEG^(5k)-Cys₄-L₈-CA₈ was as follows: (Fmoc)Lys(Dde)-OH (3eq.) was coupled onto the N terminus of PEG using DIC and HOBt ascoupling reagents until a negative Kaiser test result was obtained,thereby indicating completion of the coupling reaction. PEGylatedmolecules were precipitated by adding cold ether and then washed withcold ether twice. Fmoc groups were removed by the treatment with 20%(v/v) 4-methylpiperidine in dimethylformamide (DMF), and the PEGylatedmolecules were precipitated and washed three times by cold ether. Whitepowder precipitate was dried under vacuum and two coupling of(Fmoc)Lys(Fmoc)-OH and one coupling of (Fmoc)lys(Boc)-OH were carriedout respectively to generate a third generation of dendritic polylysineterminated with four Boc and Fmoc groups on one end of PEG. After theremoval of Boc groups with 50% (v/v) trifluoroacetic acid (TFA) indichloromethane (DCM), (Fmoc)Cys(Trt)-OH, (Fmoc)Ebes-OH and Cholic acidNHS ester were coupled step by step to the terminal end of dendriticpolylysine. The Trt groups on cysteines were removed byTFA/H₂O/ethanedithiol (EDT)/triethylsilane (TIS) (94:2.5:2.5:1, v/v)resulting in PEG^(5k)-Cys₄-L₈-CA₈ thiolated telodendrimer (FIG. 10). Thethiolated telodendrimer was recovered from the mixture by three cyclesof dissolution/reprecipitation with DMF and ether, respectively.Finally, the thiolated telodendrimer was dissolved in acetonitrile/waterand lyophilized. The PEG^(5k)-CA₈ thiol free telodendrimer wassynthesized to prepare the non-cross-linked micelles according to ourpreviously reported method. BODIPY650/665 (NIRF dye) labeledtelodendrimers were synthesized by coupling BODIPY NHS ester to theamino group of the proximal lysine between PEG and cholic acid after theremoval of 1-(4,4-dimethyl-2,6-dioxocyclohex-1-yldine)ethyl (Dde)protecting group by 2% (v/v) hydrazine in DMF.

Ellman's test was used to determine the number of cysteines conjugatedto telodendrimers by free thiol groups. After adding Ellman reagents toa standard thiol (cysteine) for 15 min, a calibration curve was preparedby plotting the absorbance at 412 nm as function of cysteineconcentrations. Based on the calibration curve, the number of cysteineson the telodendrimers was calculated from the absorbance of samples inEllman's test. The mass spectra of the telodendrimers were collected onABI 4700 MALDI TOF/TOF mass spectrometer (linear mode) usingR-cyano-4-hydroxycinnamic acid as a matrix. ¹H NMR spectra of thepolymers were recorded on an Avance 500 Nuclear Magnetic ResonanceSpectrometer using CDCl₃ and D₂O as solvents. The concentration of thepolymers was kept at 5×10⁴ M for NMR measurements.

As determined by quantitative Ellman's test by using free cysteine tocreate a standard curve, the number of covalently attached cysteines inPEG^(5k)-Cys₄-L₈-CA₈ was 3.97, which was consistent with the molecularformula of the target telodendrimer. The molecular weight ofPEG^(5k)-Cys₄-L₈-CA₈ was determined with MALDI-TOF Mass Spectrometrycomparing with the starting PEG and PEG^(5k)-CA₈. The mono-dispersedmass traces were detected for the starting PEG and the telodendrimers,and the molecular weights of the telodendrimers from MALDI-TOF MS werealmost identical to the theoretical value. The chemical shift of PEGchains (3.5-3.7 ppm) and cholic acid (0.6-2.4 ppm) could be observed inthe ¹H NMR spectra of the PEG^(5k)-Cys₄-L₈-CA₈ in CDCl₃. The integrationof these peaks can be used to calculate the chemical compositions of thetelodendrimers. The number of cholic acids determined by ¹H-NMR for thetelodendrimers was consistent with the molecular formula of the targettelodendrimers. These results demonstrate the well-defined structure oftelodendrimers. When the NMR spectrum of PEG^(5k)-Cys₄-L₈-CA₈ wasrecorded in D₂O, the cholic acid proton peaks were highly suppressed,indicating the entanglement of cholanes by the formation of core-shellmicellar structure in the aqueous environment. The CMC of PEG^(5k)-CA₈micelles and PEG^(5k)-Cys₄-L₈-CA₈ micelles before cross-linking weremeasured using pyrene as a hydrophobic fluorescent probe and found to be5.53 μM and 5.96 μM, respectively. The PEG^(5k)-Cys₄-L₈-CA₈ micellesexhibited a size of 26 nm before cross-linking, which is also similar toPEG^(5k)-CA₈ micelles. These results indicate that PEG^(5k)-CA₈ micellesand PEG^(5k)-Cys₄-L₈-CA₈ micelles have similar physical properties.

Example 2 Preparation of Disulfide Cross-Linked Micelles

20 mg PEG^(5k)-Cys₄-L₈-CA₈ telodendrimer was dissolved in 1 mL phosphatebuffered saline (PBS) to form micelles and then sonicated for 10 min.The thiol groups on the telodendrimer were oxidized to form disulfidelinkages by purging oxygen into the micelle solution. The level of freethiol groups were monitored by Ellman's test over time. The micellesolution was used for further characterizations without dialysis afterthe level of free thiol groups remained at a constant low value.

Example 3 Preparation of PTX Loaded Disulfide Cross-Linked Micelles

Loaded with Paclitaxel

PTX was loaded into the micelles by the solvent evaporation method asdescribed in our previous studies. Briefly, PTX (1, 2, 3, 5, 7.5, 9 mg)and PEG^(5k)-Cys₄-L₈-CA₈ telodendrimers (20 mg) were first dissolved inchloroform in a 10 mL round bottom flask. The chloroform was evaporatedunder vacuum to form a thin film. PBS buffer (1 mL) was added tore-hydrate the thin film, followed by 30 min of sonication. ThePTX-loaded micelles were then cross-linked via O₂-mediated oxidizationas described above. The amount of drug loaded in the micelles wasanalyzed on a HPLC system (Waters) after releasing the drugs from themicelles by adding 9 times of acetonitrile and 10 min sonication. Thedrug loading was calculated according to the calibration curve betweenthe HPLC area values and concentrations of drug standard. The loadingcapacity is defined as the highest drug concentration that can beachieved by the micelles in aqueous solution while the loadingefficiency is defined as the ratio of drug loaded into micelles to theinitial drug content. One part of the PTX-loaded micelle solutions wasstored at 4° C. for characterizations and the rest was lyophilized. ThePTX loaded non-cross-linked micelles were prepared by using PEG^(5k)-CA₈thiol free telodendrimer as reported previously.

Before cross-linking, the PTX loading capacity in PEG^(5k)-Cys₄-L₈-CA₈micelles was able to reach a level of 8.6 mg/mL (8.6 mg PTX loaded in 20mg micelles in 1 mL PBS) (FIG. 2C). The loading efficiencies were almost100% and the final particle sizes remained in the range of 25-50 nm(FIG. 2D) for all the loadings prior to cross-linking. Aftercross-linking via oxygen, the PTX loading capacity of the micellesdecreased slightly from 8.6 mg/mL to 7.1 mg/mL, which is equivalent to35.5% (w/w) of drug/micelle ratio (Table 1). It should be mentioned thatthe micelles retained the similar particle size and 100% PTX loadingefficiency at a PTX loading of 5.0 mg/mL and lower after cross-linking.However, beyond 5.0 mg/mL, the particle sizes of the cross-linkedmicelles increased (FIG. 2D) while the loading efficiency decreased to81%.

Loaded with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanineperchlorate (DiD)

DiD (hydrophobic NIRF dye) was loaded into the micelles using the samemethod as described above. The micelle solution was filtered with 0.22μm filter to sterilize the sample.

Example 4 Characterization of PTX Loaded Disulfide Cross-Linked MicellesGeneral Characterization

The size and size distribution of the micelles were measured by dynamiclight scattering (DLS) instruments (Microtrac). The micelleconcentrations were kept at 1.0 mg/mL for DLS measurements. The zetapotential of these micelles was measured by DLS using the function ofZetatrac (Microtrac). All measurements were performed at 25° C., anddata were analyzed by Microtrac FLEX Software 10.5.3. The morphology ofmicelles was observed on a Philips CM-120 transmission electronmicroscope (TEM). The aqueous micelle solution (1.0 mg/mL) was depositedonto copper grids, stained with phosphotungstic acid, and measured atroom temperature. The critical micelle concentration (CMC) of thePEG^(5k)-CA₈ micelles and PEG^(5k)-Cys₄-L₈-CA₈ micelles before and aftercross-linking was measured through fluorescence spectra by using pyreneas a hydrophobic fluorescent probe as described previously. Briefly,micelles were serially diluted in PBS to give the concentrations rangingfrom 5×10⁻⁷ to 5×10⁻⁴ M. The stock solution of pyrene in methanol wasadded into the micelle solution to make a final concentration of pyreneof 2×10⁻⁶ M. The solution was mildly shaken over night. Excitationspectra were recorded ranging from 300 to 360 nm with a fixed emissionat 390 nm. The ratios of the intensity at 337 to 332 nm from theexcitation spectra of pyrene were plotted against the concentration ofthe micelles. The CMC was determined from the threshold concentration,where the intensity ratio 1337/1332 begins to increase markedly.

The PEG^(5k)-Cys₄-L₈-CA₈ micelles without drug loading (empty micelles)were further characterized with respect to particle size, apparent CMCand zeta potential following disulfide cross-linking. After the instantformation of micelles upon dispersion in aqueous solution, the freethiol groups of PEG^(5k)-Cys₄-L₈-CA₈ were oxidized by oxygen to formdisulfide linkages. The O₂-mediated oxidization was monitored byEllman's test. The free thiol groups were oxidized over time and morethan 85% of thiol groups were reacted to form disulfide after 48 h ofoxidation. Interestingly, the PEG^(5k)-Cys₄-L₈-CA₈ micelles retained asimilar particle size of around 27 nm with the narrow distributionfollowing disulfide cross-linking. This result suggests that thedisulfide bond formation is an event that occurs within micelles. ThePEG outer corona confines the cross-linking reaction intra-micellarly,preventing the formation of inter-micellar aggregates. Aftercross-linking, the apparent CMC of PEG^(5k)-Cys₄-L₈-CA₈ micellesdecreased to 0.67 μM, which is 9 times lower than that of thenon-cross-linked micelles. This observation for the cross-linkedmicelles is consistent with the reported cross-linked Pluronic L121micelles. The zeta potential of the micelles was measured to be nearlyneutral since these micelles were composed of unchargedPEG^(5k)-Cys₄-L₈-CA₈. PEG^(5k)-CA₈ micelles were selected as absolutenon-cross-linked micelles (NCMs) in the following in vitro and in vivoevaluations, instead of the PEG^(5k)-Cys₄-L₈-CA₈ micelles prior tooxidation, because the latter will undoubtedly be partially cross-linkedby oxygen in air upon storage. Furthermore, we want to directly comparethe current cross-linked nanoformulation with our previously publishednon-cross-linked PEG^(5k)-CA₈ micelle.

TABLE 1 Physico-chemical properties of telodendrimers (PEG^(5k)-CA₈ andPEG^(5k)-Cys₄-L₈-CA₈) and the corresponding non-cross-linked anddisulfide cross-linked micelles (NCMs and DCMs). PTX loading Mw Mw CMCSize capacity Size with Telodendrimers (theo.)^(a) (MS)^(b)N_(cysteines) ^(c) N_(CA) ^(d) Micelles (μM) ^(f) (nm) ^(g) (mg/mL)^(h)PTX (nm) ^(i) PEG^(5k)-CA₈ 9059 8918 0 7.5 NCMs 5.53 22 ± 5 9.0 26 ± 4PEG^(5k)-Cys₄-L₈- 11313 11198 3.97 7.3 DCMs ^(e) 0.67 28 ± 4 7.1 27 ± 6CA₈ ^(a)Theoretical molecular weight. ^(b)Obtained via MALDI-TOF MSanalysis (linear mode). ^(c)Number of cysteines, obtained via Ellman'stest. ^(d)Number of cholic acids, calculated based on the averageintegration ratio of the peaks of methyl proton 18, 19, and 21 in cholicacid at 0.66, 0.87 and 1.01 ppm and methylene proton of PEG at 3.5-3.7ppm in ¹H-NMR spectra in CDCl_(3.) The molecular weight of the startingPEG was 4912. ^(e) Formed by PEG^(5k)-Cys₄-L₈-CA₈ micelles aftercross-linking. ^(f) Measured via fluorescent method by using pyrene as aprobe. ^(g) Particle size of NCMs and DCMs, measured by dynamic lightscattering particle sizer (Microtrac). ^(h)PTX loading capacity of NCMsand DCMs, in the presence of 20 mg/mL of telodendrimers, measured byHPLC. ^(i) Measured by dynamic light scattering particle sizer. The PTXloading of NCMs and DCMs was 5.0 mg/mL and 4.6 mg/mL, respectively.

Stability of Micelles in SDS and Human Plasma

The stability study was performed to monitor the change in particle sizeof the DCMs and NCMs in the presence of sodium dodecyl sulfate (SDS),which was reported to be able to efficiently break down polymericmicelles. An SDS solution (7.5 mg/mL) was added to aqueous solutions ofmicelles (1.5 mg/mL). The final SDS concentration was 2.5 mg/mL and themicelle concentration was kept at 1.0 mg/mL. The size and sizedistribution of the micelle solutions was monitored at predeterminedtime intervals. The stability of the micelles was also evaluated in thepresence of GSH and NAC (20 mM) together with SDS. The lyophilizedPTX-loaded micelle powder was re-hydrated with PBS and tested under thesame conditions. At the end of the stability study, the samples werefurther observed under TEM. The stability of PTX-loaded NCMs and DCMswas further studied in 50% (v/v) plasma from healthy human volunteers.The mixture was incubated at physiological body temperature (37° C.)followed by size measurements at predetermined time intervals up to 96h.

Both PTX loaded non-cross-linked micelles (PTX-NCMs) and disulfidecross-linked micelles (PTX-DCMs) have been found to be stable at 4° C.The PTX-NCMs and PTX-DCMs were incubated with 50% human plasma, and theparticle sizes of micelles were monitored by DLS over time. Both of theDCMs and NCMs micelles with similar PTX loading retained the averageparticle size around 30 nm in human plasma for 24 hours (FIG. 3).However, the PTX-DCMs still kept the uniformity and narrow distributionin size while the PTX-NCMs showed broader size distribution andpopulation of size over 100 nm, indicating the formation of aggregates(FIG. 3).

Sodium dodecyl sulfate (SDS), a strong ionic detergent, has beenreported to be able to efficiently break down polymeric micelles. Theexchange rate between polymeric micelles and unimers is accelerated bylow concentrations of SDS while at higher concentrations, the presenceof SDS micelles solubilize the amphiphilic block copolymers resulting indestabilization of the polymeric micelles. The stability of NCMs andDCMs was also tested in the presence of the reported micelle-disruptingSDS concentration of 2.5 mg/mL. The size of SDS background is below thedetection limit of DLS analysis, showing a 0.9 nm population in thespectra. After each micelle solution (1.0 mg/mL) was mixed with anaqueous solution of SDS (2.5 mg/mL), the particle size was monitored atvarious time points. The immediate disappearance of particle size signalof the NCMs reflects the distinct dynamic association-dissociationproperty of non-cross-linked micelles (FIG. 4, FIGS. 13A & B). Theconstant particle size of the DCMs under similar condition over timeindicated that such cross-linked micelles remained intact. There-hydrated lyophilized PTX-DCMs also retained the particle size ataround 26 nm in the presence of SDS (FIGS. 3E & F).

The GSH concentration inside cells (−10 mM) is known to be substantiallyhigher than the extracellular level (−2 μM). As shown in FIG. 13E, theDCMs were stable in SDS solution with a cellular exterior level of GSH(−2 μM). However, in the presence of SDS and an intracellular reductiveGSH level (10 mM), the disulfide cross-linked micelle particle sizesignal remained unchanged for 30 min until it decreased suddenly (within10 sec), indicating that rapid dissociation of the micelle when acritical number of disulfide bonds were reduced (FIG. 4A, FIG. 13F). Theresponses of PTX-NCMs and PTX-DCMs to SDS and different levels of GSHwere similar to those of empty NCMs and DCMs, respectively. We alsofound that N-acetyl cysteine (NAC) could efficiently cleave thedisulfide bonds of the DCMs, as evidenced by the complete disappearanceof particle size of DCMs after 40 min in the presence of SDS and NAC (10mM) (data not shown). The samples of DCMs and NCMs were further examinedby TEM at the end point of the stability study. It was further confirmedthat the micellar structure of NCMs was destroyed in SDS solution (FIG.4B). The TEM images also demonstrated the micellar structure of DCMswere well retained in the presence of SDS (FIG. 4C) but efficientlybroken down in the presence of SDS and 10 mM of GSH (FIG. 4D).

Cell Uptake and MIT Assay

SKOV-3 ovarian cancer cells were seeded at a density of 50000 cells perwell in eight-well tissue culture chamber slides (BD Biosciences,Bedford, Mass., USA), followed by 24 h of incubation in McCoy's 5aMedium containing 10% FBS. The medium was replaced, and DiD labeledmicelles (100 μg/mL) were added to each well. After 30 min, 1 h, 2 h and3 h, the cells were washed three times with PBS, fixed with 4%paraformaldehyde and the cell nuclei were stained with DAPI. The slideswere mounted with cover slips and observed under confocal laser scanningmicroscope (Olympus, FV1000).

SKOV-3 cells were seeded in 96-well plates at a density of 10000cells/well 24 h prior to the treatment. The cells were first treatedwith or without GSH-OEt (20 mM) for 2 h and then washed 3 times withPBS. Empty micelles and various formulations of PTX with differentdilutions were added to the plate and then incubated for 2 h. The cellswere washed with PBS and incubated for another 22 h in a humidified 37°C., 5% CO₂ incubator. MTT was added to each well and further incubatedfor 4 h. The absorbance at 570 nm and 660 nm was detected using amicro-plate ELISA reader (SpectraMax M2, Molecular Devices, USA).Untreated cells served as a control. Results were shown as the averagecell viability [(OD_(treat)−OD_(blank))/(OD_(control)−OD_(blank))×100%]of triplicate wells.

PTX-NCMs showed comparable in vitro anti-tumor effects against SKOV-3cells as Taxol® (FIG. 6B). However, PTX-DCMs were found to be lesscytotoxic than Taxol and PTX-NCMs, which was expected due to the slowerrelease of PTX within the cell culture media as well as after thecellular uptake of PTX-DCMs (FIG. 6B). Pre-incubation of cells withGSH-OEt enhances the inhibition effect of PTX-DCMs when theconcentration of PTX was higher than 10 ng/mL (FIG. 6B). In contrast,the toxicity profile of PTX-NCMs was not affected by the GSH-OEtpre-treatement. As described above, the addition of GSH-OEt increasesthe intracellular GSH concentration, and facilitates intracellular drugrelease because of the cleavage of intra-micellar disulfide bridges ofDCMs, which results in enhanced cytotoxicity.

Hemolysis Assay

The hemolysis of NCMs and DCMs was investigated using fresh citratedblood from healthy human volunteers. The red blood cells (RBCs) werecollected by centrifugation at 1000 rpm for 10 min, washed three timeswith PBS, and then brought to a final concentration of 2% in PBS. 200μl, of erythrocyte suspension was mixed with different concentrations(0.2 and 1.0 mg/mL) of NCMs and DCMs, respectively, and incubated for 4h at 37° C. in an incubator shaker. The mixtures were centrifuged at1000 rpm for 5 min, and 100 μL of supernatant of all samples wastransferred to a 96-well plate. Free hemoglobin in the supernatant wasmeasured by the absorbance at 540 nm using a micro-plate reader(SpectraMax M2, Molecular Devices, USA). RBC incubation with Triton-100(2%) and PBS were used as the positive and negative controls,respectively. The percent hemolysis of RBCs was calculated using thefollowing formula: RBCshemolysis=(OD_(sample)−OD_(negative control))/(OD_(positive control)−OD_(negative control))×100%.

As shown in FIG. 6C, empty NCMs were found to have dose dependent RBClysis. The percentage of hemolysis increased from 9.0% to 16.3% with theincreasing NCMs concentrations from 0.2 mg/mL to 1.0 mg/mL. In contrast,empty DCMs showed no observable hemolytic activities (<5%) in the RBCsat the same experimental concentrations. The intra-micellar disulfidebridges prevent DCMs from dissociation to form amphiphilictelodendrimers, thus minimizing the hemolytic activities.

In vivo Blood Elimination Kinetics and Biodistribution

DiD or BODIPY labeled NCMs and DCMs were prepared for the bloodelimination study. The concentration of BODIPY conjugated micelles was 5mg/mL. The concentration of DiD loaded micelles was 20 mg/mL with DiDloading at 0.5 mg/mL. The fluorescence spectra of these fluorescentlylabeled micelles diluted 20 times by PBS were characterized byfluorescence spectrometry (SpectraMax M2, Molecular Devices, USA). 100μL of BODIPY conjugated or DiD loaded NCMs and DCMs were injected intotumor free nude mice via tail vein. 50 μL blood was collected atdifferent time points post-injection to measure the fluorescence signalof DiD or BODIPY.

Nude mice with subcutaneous SKOV-3 tumors of an approximate 8-10 mmdiameter were subjected to in vivo NIRF optical imaging. At differenttime points post injection of DiD and PTX co-loaded cross-linkedmicelles (the concentrations of DiD and PTX were both 0.5 mg/mL), micewere scanned using a Kodak multimodal imaging system IS2000MM with anexcitation bandpass filter at 625 nm and an emission at 700 nm. The micewere anaesthetized by intraperitoneal injection of pentobarbital (60mg/kg) before each imaging. After in vivo imaging, animals wereeuthanized by CO₂ overdose at 24 h after injection. Tumors and majororgans were excised and imaged with the Kodak imaging station.

The NIRF signal of blood background was found to be very low. DiD orBODIPY 650/665 labeled NCMs and DCMs had comparable in vitro nearinfrared fluorescence signals at an estimated in vivo concentration.After i.v. injection into mice, BODIPY signal of NCMs was rapidlyeliminated from circulation and fell into the background level within 8hours post injection. It should be mentioned that BODIPY signal of DCMsin blood was 8 times higher than that of NCMs at 8 hours post injectionand sustained up to 24 hours (FIG. 7A). The overall micelleconcentrations injected for DiD loaded NCMs or DCMs were 20 mg/mL, 4times higher than that for BODIPY labeled NCMs or DCMs (5 mg/mL).Nevertheless, a similar trend of circulation kinetics was observed forthe DiD loaded NCMs and DCMs. DiD signal of NCMs decreased faster inspite of the initial increase while that of the DCMs sustained in bloodup to 30 h (FIG. 7B). The above profiles of elimination kinetics forboth vehicle and payload indicated that the cross-linked micelles havelonger blood circulation time than the non-cross-linked micelles.

Ex vivo imaging at 72 h post injection further confirmed thepreferential uptake of DCMs in tumor compared to normal organs (FIG. 8).This is due to the prolonged in vivo circulation time of the micellesand the size-mediated EPR effect.

In Vivo Toxicity

In order to investigate for telodendrimer related toxicity, both emptynon-cross-linked and cross-linked micelles were injected in tumor freenude mice at the single dose of 200 mg/kg and 400 mg/kg via tail vein.Mice were checked for possible signs of toxicity and the survivalsituation was monitored daily for two weeks.

At a single dose of 200 mg/kg, all the mice in NCMs group showedsignificant body weight loss and 1 of 4 mice died within 2 days postinjection. All the mice in the group treated with a higher NCMs dose of400 mg/kg died within 2 hours post injection. Bloody urine was observedfor some mice, indicating the hemolytic potential caused by NCMs at highdosage. On the contrary, none of the mice treated with DCMs were dead atthe single dose of 400 mg/kg and no obvious signs of toxicity wereobserved within two weeks post-injection.

Example 5 Drug Release from Disulfide Cross-Linked Micelle

PTX-loaded cross-linked micelle solution was prepared to determine thein vitro drug release profile. The initial PTX concentration was 4.6mg/mL. Aliquots of PTX-loaded cross-linked micelle solution wereinjected into dialysis cartridges (Pierce Chemical Inc.) with a 3.5 kDaMWCO. The cartridges were dialyzed against 1 L PBS with various GSHconcentrations (0, 2 μM, 1 mM, and 10 mM) at 37° C. In order to make anideal sink condition, 10 g charcoal was added in the release medium. Theconcentration of PTX remaining in the dialysis cartridge at various timepoints was measured by HPLC. The drug release profiles of Taxol® and PTXloaded non-cross-linked micelles (PTX concentration: 5.0 mg/mL) weredetermined under identical condition for comparison. In someexperiments, GSH or NAC (10 mM) were added to the release medium at aspecific release time (5 h). The PTX release profiles of the lyophilizedand rehydrated micelle solution were evaluated under the sameconditions. Values were reported as the means for each triplicatesample.

The PTX release profiles from Taxol®, NCMs and DCMs were compared byusing the dialysis method. PTX release from Taxol® was rapid and about60% of PTX was released within the first 5 h. In contrast, PTX releasefrom NCMs and DCMs was significantly slower (FIG. 14). In the presenceof GSH at its extracellular level (2 μM), the PTX release profile fromDCMs was similar to that in the release media without GSH. It was notedthat the PTX release was gradually facilitated as the GSH concentrationincreased up to the intracellular level (10 mM) (FIG. 5A). This drugrelease study also indicated that PTX release from DCMs wassignificantly slower than that from NCMs (FIG. 14, FIG. 5B). When 10 mMGSH was added at the 5 h time point, there was a burst of drug releasefrom the DCMs but not the NCMs (FIG. 5B). Subsequent release curves(after 6 h) of both preparations were identical (FIG. 5B). The PTXrelease profiles of the re-hydrated lyophilized PTX-DCMs were found tobe very similar to the fresh sample and can be greatly facilitated byGSH (FIG. 5C). NAC, a FDA approved reducing agent, was demonstrated tohave the same effect as GSH in triggering the PTX release from thedisulfide cross-linked micelles (FIG. 5D). Therefore, NAC can be appliedin vivo as an on-demand cleavage reagent via systemic i.v. injection totrigger drug release after nanotherapeutics have accumulated in tumorsites.

Example 6 Treatment of Ovarian Cancer with PTX Loaded DisulfideCross-Linked Micelles Tumor Xenograft Model

The subcutaneous xenograft model of ovarian cancer was established byinjecting 7×10⁶ SKOV-3 ovarian cells in a 100 μL of mixture of PBS andMatrigel (1:1 v/v) subcutaneously into the right flank of female nudemice.

In Vivo Therapeutic Study

Nude mice bearing SKOV-3 ovarian cancer xenografts were used to evaluatethe therapeutic efficacy of the different formulations of PTX. Thetreatments were initiated when tumor xenograft reached a tumor volume of100-200 mm³ and this day was designated as day 0. On day 0, these micewere randomly divided into seven groups and injected intravenously viathe tail vein with the formulations and repeated every 3 days for total6 doses. Injection volume was 0.1 mL for each 10 g of mouse body weight.The seven groups (n=8-10) are shown in Table 1. Taxol® was given at adose of 10 mg/kg which is close to its maximum tolerated dose (MTD). PTXloaded NCMs and DCMs were administered at the same PTX dose (10 mg/kg)for comparison. Because the micellar formulations of PTX are much moretolerated as we reported previously (MTD 75 mg/kg). The PTX loadedmicelles were also administered at a higher dose (30 mg/kg) to determineif the anti-tumor effect could be enhanced. N-acetylcysteine (NAC) is areducing agent and has been approved by FDA for mucolytic therapy (brandname: Mucomyst®) and the treatment of acetoaminophen overdose. In theseventh group, NAC was injected at a dose of 100 mg/kg into the mice viatail vein at 24 h after the administration of every dose of PTX loadedDCMs. Tumor size was measured with a digital caliper twice per week.Tumor volume was calculated by the formula (L×W²)/2, where L is thelongest and W is the shortest in tumor diameters (mm). To comparebetween groups, relative tumor volume (RTV) was calculated at eachmeasurement time point (where RTV equals the tumor volume at given timepoint divided by the tumor volume prior to initial treatment). Tomonitor potential toxicity, the body weight of each mouse was measuredevery 3 days. For humane reasons, animals were euthanized when theimplanted tumor volume reached 1500 mm³, which was considered as the endpoint of survival data.

The anti-tumor effects of PTX-DCMs and PTX-NCMs were evaluated in thesubcutaneous SKOV-3 tumor bearing mice in comparison with the clinicalformulation of PTX (Taxol®). The seven groups (n=8-10) are shown inTable 1. The tumor growth inhibition and survival rate of SKOV-3 tumorbearing mice treated with PBS, Taxol®, PTX-NCMs, and PTX-DCMs werecompared and the results are shown in FIG. 9. Compared with the controlgroup, mice in all the treatment groups showed significant inhibition oftumor growth (P<0.05). However, at the dose of 10 mg PTX/kg (MTD ofTaxol®), PTX-NCMs and PTX-DCMs exhibited superior tumor growthinhibition and longer survival time compared to Taxol®. The mediansurvival time was 21 days for PBS, 27 days for 10 mg/kg Taxol®, 28.5days for 10 mg/kg PTX-NCMs, and 32.5 days for 10 mg/kg PTX-DCMs,respectively. Importantly, the tumor growth rate of mice treated with 10mg PTX/kg PTX-DCMs was lower compared to those treated with PTX-NCMs at10 mg PTX/kg (P<0.05). PTX-DCMs showed increased in vivo therapeuticefficacy, regardless of their lower in vitro cytotoxicity against SKOV-3cancer cells, which may due to the higher amount of PTX that reached thetumor site via their prolonged circulation time.

TABLE 2 Treatment groups of nude mice bearing SKOV-3 ovarian cancerxenografts. Group Treatment^(a) Dose of PTX 1 PBS control 0 2 Taxol ® 10mg/kg 3 PTX-NCMs 10 mg/kg 4 PTX-DCMs 10 mg/kg 5 PTX-NCMs 30 mg/kg 6PTX-DCMs 30 mg/kg 7 PTX-DCMs + NAC (100 mg/kg)^(b) 30 mg/kg^(a)Administration started at the day when tumors reached a volume of100-200 mm³ and then every three days for six doses. ^(b)In group 7,N-acetylcyseine (NAC) was administrated i.v. at a dose of 100 mg/kg 24 hafter each dose of PTX-DCMs.

At the tumor site and particularly inside the tumor cells, the highglutathione level is expected to facilitate drug release from micellesand increase cytotoxicity. Three groups of mice were administrated athigher dose level: 30 mg PTX/kg PTX-NCMs, 30 mg PTX/kg PTX-DCMs and 30mg PTX/kg PTX-DCMs followed by 100 mg/kg NAC 24 h later, respectively.It is important to realize that 30 mg/kg is more than double the maximumtolerated dose (MTD) for mice if it were given in the standard CremophorEL/ethanol formuation of PTX (Taxol®. Tumor growth was inhibited in micetreated with all three micellar PTX formulations at 30 mg PTX/kg (FIG.9A). The median relative tumor volume (RTV) for all the three high dosegroups was less than 1.0 before day 36. However, tumor progression wassubsequently noted for all these high dose groups. PTX-DCMs weredemonstrated to be more efficacious in tumor inhibition than PTX-NCMsafter day 36. It should be noted that the treatment of 30 mg PTX/kgPTX-DCMs followed with 100 mg/kg of NAC was efficacious for inhibitingtumor growth. No palpable tumors were detected in 6 of the 8 mice by day93. The highest complete tumor response rate of 75% was achieved withthe combination treatment of PTX-DCMs and NAC (FIG. 9C). NAC is commonlyused in clinic as a reducing agent and has been approved by FDA formucolytic therapy and for the treatment of acetoaminophen overdose. NAChas also been employed with a number of chemotherapy agents (e.g.cisplatin, carboplatin) as a means of reducing systemic toxicity.However, in some cases, the administration of NAC also reduced thetherapeutic efficacy of these chemotherapy agents. In this study, thetreatment of 30 mg PTX/kg PTX-DCMs followed by 100 mg/kg NAC exhibited abetter anti-tumor effect than the treatment without NAC. This resultindicated that NAC mainly played the role of a reducing agent whenadministered 24 hours after nanotherapeutic treatment to cleave theintra-micellar disulfide bridges and release the drug on-demand. Thisimportant in vivo observation has great translational potential and canbe easily tested in clinical trials in the future.

Toxicities were assessed by analyzing effects on animal behavior andbody weight change. The group of mice treated with 10 mg PTX/kg Taxol®frequently demonstrated decreased overall activity over 10 min postinjection. This is likely due to the use of Cremophor EL and ethanol asvehicle for paclitaxel and the rapid high peak level of PTX in theblood. No noticeable change in activity was observed afteradministration of 10 mg PTX/kg PTX-NCMs and 10 mg PTX/kg PTX-DCMs. Thegroup of mice receiving 30 mg PTX/kg PTX-DCMs followed with 100 mg/kg ofNAC exhibited slight more body weight loss (12.2%) during the treatmentcycle compared to other micellar PTX groups. Without being bound to anyparticular theory, one possible reason is the injection of high doseNAC. On the other hand, there may be still a small portion of PTX-DCMsin the blood circulation at 24 h post-injection. The administration ofNAC may trigger the release of drug from circulating PTX-DCMs into bloodstream, therefore resulting in toxicity to the mice. The systemictoxicity can probably be minimized by optimizing the dose and injectiontime of NAC.

Example 7 Preparation of Vincristine Loaded Disulfide Cross-LinkedMicelles

1 mg of vincristine sulfate powder was dissolved with 3 molarequivalents of triethylamine (TEA) in chloroform and mixed with 20 mgPEG^(5k)-Cys₄-L₈-CA₈ telodendrimers in a 10 mL round bottom flask.Chloroform was evaporated under vacuum to form a thin film followed byhydration of the film with 1 mL PBS buffer and 30 min of sonication. TheVCR-loaded micelles were then cross-linked via O₂-mediated oxidation asdescribed above. 1 mg of vincristine sulfate powder was dissolved with 3molar equivalents of triethylamine (TEA) in chloroform and mixed with 20mg PEG^(5k)-Cys₄-L₈-CA₈ telodendrimers in a 10 mL round bottom flask.Chloroform was evaporated under vacuum to form a thin film followed byhydration of the film with 1 mL PBS buffer and 30 min of sonication. TheVCR-loaded micelles were then cross-linked via O₂-mediated oxidation asdescribed previously. The level of free thiol groups was monitored byEllman's test over time.

Example 8 Characterization of Vincristine Loaded Disulfide Cross-LinkedMicelles

Characterization of the vincristine loaded disulfide cross-linkedmicelles was carried out using the methods described above, except whereotherwise indicated.

Stability

The stability of NCM-VCR and DCM-VCR in physiological and severe micelledisrupting conditions was compared. Both types of micelles wereincubated with 50% human plasma at 37° C. and their size was monitoredover time using DLS. After 24 h, the size of NCM-VCR increased slightlyfrom 16 to 31 nm and the particle size distribution was broadened withsome particles as large as 100 nm (FIG. 16A). In comparison, the size ofDCM-VCR was unchanged during the 24 h period (FIG. 16C). To furtherexamine micelle stability, we monitored particle size after incubatingNCM-VCR and DCM-VCR with 2.5 mg/mL SDS which is known to efficientlybreak down polymeric micelles. Immediately upon addition of SDS toNCM-VCR, micelle size was reduced from 15 to 1 nm, indicating completemicelle disruption (FIG. 16B). DCM-VCR resisted micelle disruption andmaintained particle size (FIG. 16D). The intra-micellar disulfide bondswhich stabilize DCM-VCR are reversible which allows for drug releasewhen inside the highly reducing environment of the target cell or uponexternal addition of a reducing agent such as N-acetylcysteine (NAC). Inthe presence of SDS and NAC (20 mM), the integrity of DCM-VCR wascompromised and full disruption of the particle was observed after 1 h(FIG. 16E).

Drug Release

The in vitro drug release profiles for the different formulations of VCRwere measured using the dialysis method. NCMs and DCMs were preparedwith 20 mg/mL telodendrimer and loaded with 1 mg/mL VCR. Aliquots of theconventional formulation of VCR (1 mg/mL), NCM-VCR and DCM-VCR wereinjected into dialysis cartridges with a molecular-weight cutoff (MWCO)of 3.5 kDa (Thermo Scientific, Rockford, Ill.). Cartridges were dialyzedagainst 1 L PBS at 37° C. with shaking at 100 rpm in the presence of 10g/L activated charcoal to create a sink condition. The concentration ofVCR remaining in the dialysis cartridge at various time points wasmeasured by spectrophotometry after releasing the drug from the micellesby adding 9 volumes of DMSO and 10 minutes of sonication. Values werereported as the means for each triplicate sample.

After 8 h, the conventional VCR sample had released 73% VCR compared to44% and 24% for NCM-VCR and DCM-VCR, respectively. By 24 h, almost 100%of the conventional VCR had been released while NCM-VCR and DCM-VCRretained 20% and 43% of their original VCR content. We have previouslydemonstrated that when a reducing agent such as NAC or glutathione (GSH)is added to the dialysate, drug release can be rapidly facilitated.

In Vitro Toxicity

Raji cells were seeded in 96-well plates at a density of 10,000cells/well. The cells were treated with the conventional formulation ofVCR, NCM-VCR and DCM-VCR continuously for 72 h. In a separateexperiment, Raji cells were incubated with the VCR formulations for 2 h,washed 3 times with PBS, resuspended in media and incubated for anadditional 70 h. After 72 h, cell viability was assessed using theCellTiter 96 AQueous One Solution Cell Proliferation Assay according tothe manufacturer's instructions. MTS solution (20 uL) was added to eachwell and cell viability assessed after a 1 h incubation. Cell viabilityas a percent of the untreated control for triplicate wells wascalculated as follows:

[(OD₄₉₀treated−OD₄₉₀background)/(OD₄₉₀control−OD₄₉₀background)*100].

Between the two VCR micelle formulations, NCM-VCR was more cytotoxicthan DCM-VCR. Conventional VCR at a concentration of 25 nM killed 68% ofcells while the same concentration of NCM-VCR and DCM-VCR killed 58% and36% of cells, respectively.

Example 9 Treatment of B-Cell Lymphoma with Vincristine Loaded DisulfideCross-Linked Micelles Xenograft Model

The Burkitt's B-cell lymphoma cell line, Raji, was purchased from theAmerican Type Culture Collection (ATCC; Manassas, Va., USA). Cells werecultured in ATCC formulated RPMI-1640 medium supplemented with 10% fetalbovine serum (FBS), 100 U/mL penicillin G and 100 μg/mL streptomycin at37° C. using a humidified 5% CO₂ incubator. 3 days before tumor cellimplantation, mice received 400 rads of whole body radiation. Toestablish tumors, 5×10⁶ Raji cells resuspended in PBS weresubcutaneously implanted on to the flank of each mouse.

In Vivo Therapeutic Study

NHL Raji tumors were allowed to grow until they reached a range of100-200 mm³ and this was designated as day 0. Mice were then randomlyseparated into 5 treatment groups (n=6-8 per group). The first treatmentgroup consisted of PBS as a control. The second and third groupsconsisted of the conventional formulation of VCR and DCM-VCR, both at adose of 1 mg/kg. The fourth group consisted of DCM-VCR (1 mg/kg) plusN-acetylcysteine (NAC) at 100 mg/kg. NAC is a reducing agent and hasbeen approved by FDA for mucolytic therapy (Mucomyst®) and the treatmentof acetaminophen overdose. NAC was given intravenously 24 h after theadministration of DCM-VCR. The fifth group consisted of DCM-VCR alone ata dose of 2.5 mg/kg. Treatments were administered on days 0 and 9 andwere injected through the tail vein. Body weight and tumor volume weremeasured twice per week with tumor volume assessed using digitalcalipers and calculated using the equation: (L×W²)/2. Mice weresacrificed when tumor volume exceeded 1500 mm³ or 20 mm in eitherdimension.

All VCR treatments caused a (p<0.05) reduction in tumor growth ascompared to the PBS control. Mice receiving 1 mg/kg DCM-VCR did notexhibit a superior anti-tumor effect compared with conventional VCR atthe same dose. However, mice receiving 1 mg/kg DCM-VCR followed by 100mg/kg NAC 24 h later, did exhibit a greater reduction in tumor volumecompared with conventional VCR at the same dose (p<0.05). We attributethe greater efficacy to the on-demand drug release from DCM-VCR once itaccumulated at the tumor site. Although 1 mg/kg conventional VCR andDCM-VCR without NAC exhibited equivalent efficacy, the group of micereceiving DCM-VCR (with and without NAC) lost significantly less weightthan the group of mice receiving conventional VCR (FIG. 19B). In termsof a clinical benefit, DCM-VCR may offer a less toxic treatment optionthat does not sacrifice efficacy. The greatest reduction in tumor volumewas observed in the group receiving 2.5 mg/kg DCM-VCR which exhibited asignificantly greater tumor reduction than all treatment groups(p<0.005). It should be noted that the 2.5 mg/kg DCM-VCR treatment grouplost an equivalent amount of weight as the 1 mg/kg conventional VCRgroup (FIG. 19B).

TABLE 3 Eight days after the final treatment, the blood of mice (n = 3)from each group was collected for determination of complete blood count.WBC RBC (K/uL) (M/uL) Hemoglobin (g/dL) Platelets (K/uL) PBS 7.26 ± 2.3110.17 ± 0.18  15.6 ± 0.24 695.3 ± 144.2 Conventional VCR 1 mg/kg 5.69 ±1.51 9.58 ± 0.80 15.2 ± 0.60   971 ± 103.3 DCM-VCR 1 mg/kg 5.65 ± 1.609.30 ± 0.43 14.8 ± 0.51   876 ± 162.4 DCM-VCR 1 mg/kg + 7.01 ± 1.67 9.98± 0.95 15.5 ± 1.25 811.7 ± 232.2 N-Ac 100 mg/kg DCM-VCR 2.5 mg/kg 6.45 ±1.22 8.95 ± 0.43 14.8 ± 0.81  1268 ± 206.9

TABLE 4 Eight days after the final treatment, the blood of mice (n = 3)from the PBS, conventional VCR 1 mg/kg and DCM-VCR 2.5 mg/kg groups wascollected for serum chemistry analysis including alanineaminotransferase (ALT), aspartate aminotransferase (AST), totalbilirubin, blood urea nitrogen (BUN) and creatinine. ALT AST CreatinineTotal Bilirubin (K/uL) (M/uL) BUN (K/uL) (K/uL) (g/dL) PBS 38.5 ± 3.35109.4 ± 6.5  25.7 ± 2.6 .149 ± .01 .128 ± .006 Conventionl VCR 1 mg/kg33.6 ± 1.06 101.1 ± 19.6 32.2 ± 3.8 .202 ± .02  .12 ± .017 DCM-VCR 2.5mg/kg 31.9 ± 6.92   91 ± 7.8 33.1 ± 2.9 .174 ± .02 .089 ± .008

Eight days after the final treatment we dissected the sciatic nerve frommice of the PBS, conventional VCR (1 mg/kg) and DCM-VCR (2.5 mg/kg)groups to assess the acute neurotoxic effects of VCR by histologicalanalysis. VCR is known to cause axonal degeneration and demyelination ofnerve fibers which can be observed with either light or electronmicroscopy. Histological analysis revealed no obvious damage to thenerve fibers from the groups treated with conventional VCR (1 mg/kg) andDCM-VCR (2.5 mg/kg) and no differences compared to the PBS control group(FIG. 20).

Maximum Tolerated Dose

The maximum tolerated dose of the conventional formulation of VCR andDCM-VCR was investigated in healthy female balb/c mice. Mice (n=4) weretreated with the conventional formulation of VCR or DCM-VCR at doses of1.5, 2.5, 3.5 and 4.5 mg VCR/kg. VCR was administered through the tailvein every 10 days for a total of two treatments. Body weight and othersymptoms of toxicity (unkempt fur, ataxia, piloerection, hind limbparalysis) were observed daily for 20 days. The MTD was defined as theallowance of a median body weight loss of 20% and causes neither deathdue to toxic effects nor remarkable changes in the general signs within2 weeks after administration.

Mice treated with 1.5 mg/kg of conventional VCR had significant weightloss (18%). After day 4, these mice started to regain weight and reachedtheir initial weight on day 10. The mice treated with doses higher than1.5 mg/kg of conventional VCR lost >20% of their initial weight and weresacrificed. The MTD of conventional VCR determined in this study wassimilar to the MTD observed previously by other groups. Mice treatedwith the same doses of VCR in the form of DCM-VCR exhibitedsignificantly less weight loss at all doses. Only the group of micereceiving the highest dose of 4.5 mg/kg DCM-VCR lost >20% of theirinitial weight and were sacrificed. Thus, DCM-VCR was able to increasethe MTD of VCR from 1.5 to 3.5 mg/kg (>2-fold). Dose intensification ofVCR in a clinical setting is significant as it may allow patients toreceive a full dose of chemotherapy without the limiting toxicities.

Toxicity

Mice from the above mentioned therapeutic study were also used toinvestigate VCR hematologic and neurotoxicity. Eight days after thefinal treatment, the blood of mice (n=3) from each group was collectedfor determination of complete blood count as well as analysis of serumchemistry including alanine aminotransferase (ALT), aspartateaminotransferase (AST), total bilirubin, blood urea nitrogen (BUN) andcreatinine. To compare the neurotoxic effects of the vincristineformulations, mice (n=3) were sacrificed eight days after the finaltreatment and the sciatic nerve was carefully dissected from theproximal aspect of the thigh to the knee joint proximal to its point ofdivision into common peroneal, tibial, and sural nerves. The nerves wereprocessed into epoxy blocks and 500 nm sections were cut, collected ontoslides and stained with Methylene Blue and Azure B stain. Sections wereimaged on an Olympus BH-2 microscope and images acquired using a SpotInsight digital camera (Diagnostic Instruments, Inc.).

Example 10 Preparation of Catechol Modified Conjugates(PEG^(5k)-Catechol₂-CA₈ and PEG^(5k)-Catechol₄-CA₈)

The telodendrimers containing two and four 3,4-Dihydroxybenzoic acids(named as PEG^(5k)-Catechol₂-CA₈ and PEG^(5k)-Catechol₄-CA₈,respectively) were synthesized via solution-phase condensation reactionsfrom MeO-PEG-NH₂ via stepwise peptide chemistry. The typical procedurefor synthesis of PEG^(5k)-Catechol₂-CA₈ and PEG^(5k)-Catechol₄-CA₈ wasas follows: (Fmoc)Lys(Boc)-OH (3 eq.) was coupled onto the N terminus ofPEG using DIC and HOBt as coupling reagents until a negative Kaiser testresult was obtained, thereby indicating completion of the couplingreaction. PEGylated molecules were precipitated by adding cold ether andthen washed with cold ether twice. Fmoc groups were removed by thetreatment with 20% (v/v) 4-methylpiperidine in dimethylformamide (DMF),and the PEGylated molecules were precipitated and washed three times bycold ether. White powder precipitate was dried under vacuum and onecoupling of (Fmoc)Lys(Boc)-OH and three couplings of (Fmoc)lys(Fmoc)-OHwere carried out respectively to generate a third generation ofdendritic polylysine terminated with eight Fmoc groups on one end ofPEG. Cholic acid NHS ester were then coupled to the terminal end ofdendritic polylysine. (Fmoc)Ebes-COOH was coupled to the amino groups ofthe proximal lysines between PEG and cholic acid upon the removal of Bocgroups with 50% (v/v) trifluoroacetic acid (TFA) in dichloromethane(DCM). After the removal of Fmoc groups, one part of the polymer wascoupled with 3,4-Dihydroxybenzoic acid resulting inPEG^(5k)-L₂-Catechol₂-CA₈ telodendrimer (Scheme S-1). The other part ofthe polymer was coupled with (Fmoc)lys(Fmoc)-OH and 3,4-Dihydroxybenzoicacid subsequently to generate PEG^(5k)-Catechol₄-CA₈ telodendrimer(Scheme S-1).

In order to prepare Rhodamine B labeled telodendrimers,(Fmoc)Lys(Dde)-OH was coupled to MeO-PEG-NH₂ initially to introduce an1-(4,4-dimethyl-2,6-dioxocyclohex-1-yldine)ethyl (Dde) protected aminogroup. Rhodamine β isothiocyanate was conjugated to the amino group ofthe proximal lysine between PEG and cholic acids in the finaltelodendrimers after the removal of Dde protecting group by 2% (v/v)hydrazine in DMF.

Example 11 Preparation of 4-Carboxyphenyl Boronic Acid ModifiedConjugates (PEG^(5k)-BA₂-CA₈ and 3-Carboxy-5-Nitrophenyl Boronic AcidModified Conjugates (PEG^(5k)-NBA₂-CA₈ and PEG^(5k)-NBA₄-CA₈)

The telodendrimers containing two or four 4-Carboxyphenylboronic acidand 3-Carboxy-5-nitrophenylboronic acids (named as PEG^(5k)-BA₂-CA₈,PEG^(5k)-BA₄-CA₈, PEG^(5k)-NBA₂-CA₈ and PEG^(5k)-NBA₄-CA₈, respectively)were synthesized via the similar strategy as described above.4-Carboxyphenylboronic acid pinacol ester and3-Carboxy-5-nitrophenylboronic acid pinacol ester were coupled to theEbes linkers or lysines between PEG and cholic acids at the last step.The four kinds of boronic acid containing telodendrimers were generatedupon the removal of the pinacol esters with 50% (v/v) TFA in DCM. Thetelodendrimers was recovered from the mixture by three cycles ofdissolution/reprecipitation with DMF and ether, respectively. Finally,the telodendrimers were dissolved in acetonitrile/water and lyophilized.The PEG^(5k)-CA₈ parent telodendrimerwas synthesized to prepare thenon-cross-linked micelles according to our previously reported method.

Example 12 Preparation of Boronate Cross-Linked Micelles

Two distinct boronic acid-containing telodendrimer andcatechol-containing telodendrimer (total 20 mg) were first dissolved inanhydrous chloroform in a 10 mL round bottom flask. The chloroform wasevaporated under vacuum to form a thin film. PBS buffer (1 mL) was addedto re-hydrate the thin film, followed by 30 min of sonication. Boronateester bonds formed between boronic acids and catechols of adjacenttelodendrimers, upon self-assembly in PBS, resulted in the formation ofboronate cross-linked micelles (BCM). The micelle solution was filteredwith 0.22 μm filter to sterilize the sample.

Example 13 Preparation of Drug Loaded Boronate Cross-Linked Micelles

Hydrophobic anti-cancer drug, such as paclitaxel (PTX), doxorubicin(DOX) and vincristine (VCR), were loaded into the micelles by thesolvent evaporation method as described in our previous studies.Briefly, drug (2.0 mg) and telodendrimers (total 20 mg) were firstdissolved in anhydrous chloroform in a 10 mL round bottom flask. Thechloroform was evaporated under vacuum to form a thin film. PBS buffer(1 mL) was added to re-hydrate the thin film, followed by 30 min ofsonication. The unloaded PTX was removed by running the micellesolutions through centrifugal filter devices (MWCO: 3.5 kDa, Microcon®).The PTX loaded micelles on the filters were recovered with PBS. Theamount of drug loaded in the micelles was analyzed on a HPLC system(Waters) after releasing the drugs from the micelles by adding 9 timesof acetonitrile and 10 min sonication. The drug loading was calculatedaccording to the calibration curve between the HPLC area values andconcentrations of drug standard. The loading efficiency is defined asthe ratio of drug loaded into micelles to the initial drug content.Hydrophobic dye (DiO or DiD) was loaded into the micelles using the samestrategy. The amount of dye loaded in the micelles was analyzed on afluorescence spectrometry (SpectraMax M2, Molecular Devices, USA) afterreleasing the drugs from the micelles by adding 9 times of acetonitrileand 10 min sonication. The dye loading was calculated according to thecalibration curve between the fluorescence intensity and concentrationsof dye standard in acetonitrile.

Example 14 Characterization of Drug Loaded Boronate Cross-LinkedMicelles General Characterization

The size and size distribution of the micelles were measured by dynamiclight scattering (DLS) instruments (Microtrac). The micelleconcentrations were kept at 1.0 mg/mL for DLS measurements. Allmeasurements were performed at 25° C., and data were analyzed byMicrotrac FLEX Software 10.5.3. The morphology of micelles was observedon a Philips CM-120 transmission electron microscope (TEM). The aqueousmicelle solution (1.0 mg/mL) was deposited onto copper grids, stainedwith phosphotungstic acid, and measured at room temperature. Theapparent critical micelle concentration (CMC) of the NCM and BCMs wasmeasured through fluorescence spectra by using pyrene as a hydrophobicfluorescent probe as described previously. Briefly, micelles wereserially diluted in PBS to give the concentrations ranging from 5×10⁻⁷to 5×10⁻⁴ M. The stock solution of pyrene in methanol was added into themicelle solution to make a final concentration of pyrene of 2×10⁻⁶ M.The solution was mildly shaken over night. Excitation spectra wererecorded ranging from 300 to 360 nm with a fixed emission at 390 nm. Theratios of the intensity at 337 to 332 nm from the excitation spectra ofpyrene were plotted against the concentration of the micelles. The CMCwas determined from the threshold concentration, where the intensityratio 1337/1332 begins to increase markedly.

TABLE 5 Characterization of boronate cross-linked micelles andnon-cross-linked micelles. Micelle Size CMC Stability PTX FormulationTelodendrimer pair (nm)^([a]) (μg/mL)^([b]) In SDS^([c]) Content^([d])BCM1 PEG^(5k)-BA₂-CA₈, 23 ± 4 10.5 2 min 9.9% PEG^(5k)-Catechol₂-CA₈BCM2 PEG^(5k)-NBA₂-CA₈, 26 ± 6 8.7 30 min 9.8% PEG^(5k)-Catechol₂-CA₈BCM3 PEG^(5k)-BA₄-CA₈, 22 ± 3 7.4 5 min 9.8% PEG^(5k)-Catechol₄-CA₈ BCM4PEG^(5k)-NBA₄-CA₈, 27 ± 5 4.2 Long term 9.9% PEG^(5k)-Catechol₄-CA₈ NCMPEG^(5k)-CA₈ 22 ± 6 50.1 <10 sec 10.0% ^([a])Measured by dynamic lightscattering (DLS). ^([b])Measured via fluorescent method by using pyreneas a probe. ^([c])The total period of time that the micelles retainedtheir sizes in SDS, continuously measured by DLS every 10 sec at pH 7.4.^([d])PTX loading content of micelles (drug/polymer, w/w), in thepresence of 20 mg/mL of total telodendrimers and 2.0 mg/mL PTX initialloading, measured by HPLC.

TABLE 6 Characterization of the telodendrimers. N_(BA) ^(g) N_(NBA) ^(h)Mw Mw N_(CA) ^(c) N_(BA) ^(e) N_(NBA) ^(f) N_(catechol) ^(d) (ARS (ARSTelodendrimers (theo.)^(a) (MS)^(b) (NMR) (NMR) (NMR) (NMR) assay)assay) PEG^(5k)-CA₈ 9059 8918 7.5 — — — — — PEG^(5k)-Catechol₂- 98939928 8.1 — — 2.1 — — CA₈ PEG^(5k)-Catechol₄- 10419 10376 7.6 — — 4.1 — —CA₈ PEG^(5k)-BA₂-CA₈ 9917 9801 7.6 1.7 — — 2.5 — PEG^(5k)-BA₄-CA₈ 1046710393 7.6 3.8 — — 4.2 — PEG^(5k)-NBA₂-CA₈ 9971 9837 7.7 — 1.8 — — 2.3PEG^(5k)-NBA₄-CA₈ 10650 10278 7.5 — 4.0 — — 4.1 ^(a)Theoreticalmolecular weight. ^(b)Obtained via MALDI-TOF MS analysis (linear mode).The boronic acid containing telodendrimers were measured in the pinacolester form and the molecular weight was calculated as described above.^(c)Number of cholic acids, calculated based on the average integrationratio of the peaks of methyl proton 18, 19, and 21 in cholic acid at0.66, 0.87 and 1.01 ppm and methylene proton of PEG at 3.5-3.7 ppmin¹H-NMR spectra in DMSO-d6 (90° pulse). The molecular weight of thestarting PEG was 4912. ^(d)Number of phenylboronic acids, calculatedbased on the average integration ratio of the peaks of the phenylprotons of phenylboronic acids (6.7-7.2 ppm) and methylene proton of PEGto in¹H-NMR spectra in DMSO-d6 (90° pulse). ^(e)Number ofnitro-phenylboronic acids, calculated based on the average integrationratio of the peaks of the phenyl protons of nitro-phenylboronic acids(8.6-8.9 ppm) and methylene proton of PEG to in¹H-NMR spectra in DMSO-d6(90° pulse). ^(f)Number of 3,4-Dihydroxybenzoic acids (catechols),calculated based on the average integration ratio of the peaks of thephenyl protons of catechols (6.7-7.5 ppm) and methylene proton of PEG toin¹H-NMR spectra in DMSO-d6 (90° pulse). ^(g)Number of phenylboronicacids, determined by ARS colorimetric assay. ^(h)Number ofnitro-phenylboronic acids, determined by ARS colorimetric assay.

ARS Based Colorimetric and Fluorescence Assay

ARS is a catechol dye displaying dramatic changes in color andfluorescence intensity upon binding to boronic acid. In this study, weutilized ARS indicator based colorimetric assay to estimate theconcentration of boronic acid on telodendrimers. Briefly, as theconcentration of boronic acid is increased, a visible color change fromburgundy to yellow was observed. The color and absorbance changes of ARSwere observed with adding 3-Carboxy-5-nitrophenylboronic acid. Theabsorbance of the free ARS at 520 nm decreases as boronic acids added,and a new absorbance at 460 nm appears. A calibration curve was preparedby plotting the absorbance changes at 460 nm (ΔA) as function ofconcentrations of 4-Carboxyphenylboronic acid ([BA]) and3-Carboxy-5-nitrophenylboronic acid ([NBA]). Based on the calibrationcurve, the number of boronic acids on the telodendrimers was calculatedfrom the absorbance of samples in the colorimetric assay (Table 6).

ARS also displays a dramatic change in fluorescence intensity inresponse to the binding of boronic acids. Boronic acid containingtelodendrimers solutions (boronic acid concentrations: 0-5 mM) weremixed ARS solution in PBS at pH 7.4 and the fluorescence signal of themixtures was measured by fluorescence spectrometry (Nanodrop3000,Microtrac). The final concentration of ARS was fixed at 0.1 mM. ARSfluorescence assay was further used to characterize the binding betweenthe boronic acid containing telodendrimers and catechol containingtelodendrimers. In this experiment, the final concentration of ARS andboronic acid of boronic acid containing telodendrimers were fixed at 0.1mM. Different molar ratio of catechol containing telodendrimers waspremixed with boronic acid containing telodendrimers (0.1 mM) inanhydrous chloroform. The chloroform was evaporated and the thin film onthe inner surface of flask was re-hydrated with PBS buffer to generateboronate crosslinked micelles. ARS solution was then mixed with theabove micelle solutions and the fluorescence signal of the mixtures wasmeasured by fluorescence spectrometry (Nanodrop3000, Microtrac).

When the concentrations of ARS and boronic acid containingtelodendrimers were fixed at 0.1 mM, the fluorescence of ARS wasdramatically suppressed with increasing amounts ofPEG^(5k)-CatechoL-CA₈(0 to 0.5 mM) (FIG. 22). These results are aqualitative indication of the formation of catechol-boronatecrosslinking esters as ARS was prevented from complexation with boronicacid containing telodendrimers.

Stability

The stability study was performed to monitor the change in particle sizeof the NCM and BCMs in the presence of sodium dodecyl sulfate (SDS),which was reported to be able to efficiently break down polymericmicelles. An SDS solution (7.5 mg/mL) was added to aqueous solutions ofmicelles (1.5 mg/mL). The final SDS concentration was 2.5 mg/mL and themicelle concentration was kept at 1.0 mg/mL. The size and sizedistribution of the micelle solutions was monitored continuously viadynamic light scattering (DLS) instruments (Microtrac) for 2 days. Thestability of the micelles was also evaluated in PBS at different pHlevels or in presence of mannitol and glucose (0, 10 mM, 50 mM, and 100mM), together with SDS. Hydrogen chloride and sodium hydroxide solutionswere used to prepare PBS at different pH levels. The pH values of thebuffer were determined by a digital pH meter (Φ350 pH/Temp/mV meter,Beckman Coulter, USA) which gave pH values within 0.01 units. During thestability study, a small portion of the samples were taken out andfurther observed under TEM. The stability of NCM and BCMs was furtherstudied in 50% (v/v) plasma from healthy human volunteers. The mixturewas incubated at physiological body temperature (37° C.) followed bysize measurements at predetermined time intervals up to 96 h.

The rapid disappearance (<10 sec) of the particle size signal for theNCM reflects the loss of integrity (FIGS. 30A & C). The BCM 1, BCM2 andBCM3 retained the size in SDS for 2 min, 5 min and 30 min, respectively(Table 5). Despite an initial decrease, the constant particle size wasobserved over 2 days for BCM4 treated under the same conditionsindicating that the cross-linked micelles self-assembled from thetelodendrimer pair of PEG^(5k)-NBA₄-CA₈ and PEG^(5k)-Catechol₄-CA₈remained intact (FIG. 22B, FIG. 30). BCM3 and BCM4, containing doublethe number of boronate esters retained their structural integritysignificantly longer in the presence of SDS, when compared to BCM1 andBCM2, respectively. BCM2 and BCM4 crosslinked via nitro phenyl boronateesters were more stable than the corresponding phenyl boronate esterscrosslinked micelles BCM1 and BCM3.

We further investigated the response to pH- and diol- for BCM4 in thepresence of SDS. The particle size signal of BCM4 decreased suddenly(within 2 min) in SDS after 120 min incubation in pH 5.0, indicatingthat the micelle rapidly dissociated when a critical percentage ofboronate bonds were hydrolyzed (FIG. 22B, FIG. 30G). We found thatmannitol (containing three cis-diol pairs) could also efficiently cleavethe crosslinking boronate bonds of the BCM4, as evidenced by the rapidreduction in particle size of BCM4 in the presence of SDS and excess ofmannitol (100 mM) (FIG. 22A, FIG. 30H). On the contrary, the size ofBCM4 persisted in the presence of both SDS and 100 mM glucose(containing one cis-diol) (FIG. 30I). TEM permitted the confirmationthat the micellar structure of NCM was disrupted in SDS solution. TheTEM graphs also demonstrated the micellar structure of BCM4 was wellretained in SDS at pH 7.4 (FIG. 22C2) but was rapidly disrupted in SDSat pH 5.0 or in the presence of 100 mM mannitol (FIG. 22C3, C4).

Cell Uptake and MTT Assay

SKOV-3 ovarian cancer cells were seeded at a density of 50000 cells perwell in eight-well tissue culture chamber slides (BD Biosciences,Bedford, Mass., USA), followed by 24 h of incubation in McCoy's 5aMedium containing 10% FBS. The medium was replaced, and DiD labeledmicelles (100 μg/mL) were added to each well. After 30 min, 1 h, 2 h and3 h, the cells were washed three times with PBS, fixed with 4%paraformaldehyde and the cell nuclei were stained with DAPI. The slideswere mounted with cover slips and observed under confocal laser scanningmicroscope (Olympus, FV1000). For the DiD channel, the excitation wasset to 625 nm while the emission was set to 700 nm.

SKOV-3 ovarian cancer cells were seeded in 96-well plates at a densityof 5000 cells/well 24 h prior to the treatment. The culture medium wasreplaced with fresh medium containing various formulations of PTX withdifferent dilutions at pH 7.4 or 5.0, in the absence or in the presenceof 100 mM mannitol. The cells were washed with PBS and incubated foranother 23 h in a humidified 37° C., 5% CO₂ incubator. MIT was added toeach well and further incubated for 4 h. The absorbance at 570 nm and660 nm was detected using a micro-plate ELISA reader (SpectraMax M2,Molecular Devices, USA). Untreated cells served as a control. Resultswere shown as the average cell viability[(OD_(treat)−OD_(blank))/(OD_(control)−OD_(blank))×100%] of triplicatewells. The cells were also treated with telodendrimers and emptycrosslinked micelles with different dilutions and incubated for total 72h in order to evaluate telodendrimer related toxicity.

PTX-NCM showed comparable in vitro anti-tumor effects against SKOV-3cells as Taxol® (free drug of paclitaxel). PTX-BCM4 was found to beconsiderably less cytotoxic than Taxol® and PTX-NCM at equal doselevels. There were minimal changes in the toxicity profile of PTX-NCMand free drug triggered with acidic pH and mannitol. PTX-BCM4 showedsignificantly enhanced cancer cell inhibition at pH 5.0 in the presenceof mannitol (100 mM).

In Vivo Blood Elimination Kinetics and Biodistribution

Rhodamine B labeled NCM and BCMs were prepared for the blood eliminationstudy. The concentration of rhodamine B conjugated micelles was 2.0mg/mL. The absorbance and fluorescence spectra of these micelles diluted20 times by PBS were characterized by fluorescence spectrometry(SpectraMax M2, Molecular Devices, USA). 100 μL of Rhodamine Bconjugated NCM and BCMs were injected into tumor free nude mice via tailvein. 50 μL blood was collected at different time points post-injectionto measure the fluorescence signal of Rhodamine B.

After intravenous injection into mice, rhodamine B signal of NCM wasrapidly eliminated from blood circulation and fell into the backgroundlevel within 10 hr post injection (FIG. 24F). Rhodamine B signal of BCM4in blood was 6 times higher than that of NCM at 10 hr post injection andsustained for more than 24 hr.

In Vivo Toxicity

PTX loaded NCM have been safely applied for in vivo cancer treatment.The single treatment MTD in mice was observed to be 75 mg PTX/kg, thecorresponding telodendrimer dosage was 200 mg/kg. However, without theencapsulation of hydrophobic PTX inside NCM to keep the telodendrimerstogether, the micelles tend to be more dynamic and dissociate moreeasily upon dilution, which may cause hemolytic side-effect. In order toinvestigate for telodendrimer related toxicity in vivo, both emptynon-cross-linked and cross-linked micelles were injected in tumor freenude mice at the single dose of 200 mg/kg via tail vein. PBS wasinjected into the mice as a control. Mice were checked for possiblesigns of toxicity and the survival situation was monitored daily for twoweeks. At day 7 after injection, blood samples were obtained from allthe mice for the measurement of blood cell counts, serum chemistryincluding alanine aminotransferase (ALT), aspartate aminotransferase(AST) and blood urea nitrogen (BUN).

Example 15 Release of Drug from Boronate Cross-Linked Micelle

PTX-loaded NCM and BCMs was prepared to determine the in vitro releaseprofile. The PTX loading for NCM, BCM1, BCM2, BCM3 and BCM4 were 9.9%,9.8%, 9.8%, 9.9%, 10.0% (w/w, PTX/micelle) in the presence of total 20mg telodendemers measured. Aliquots of PTX-loaded micelle solution wereinjected into dialysis cartridges (Pierce Chemical Inc.) with a 3.5 kDaMWCO. In order to make an ideal sink condition, 10 g charcoal was addedin the release medium. The cartridges were dialyzed against PBS atdifferent pH levels (pH6.5, pH6.0, pH5.5 and pH5.0) or in the presenceof various concentrations of glucose or mannitol (0, 10 mM, 50 mM, and100 mM) at 37° C. The release medium was stirred at a speed of 100 rpm.The concentration of PTX remaining in the dialysis cartridge at varioustime points was measured by HPLC. In some experiments, the releasemedium (pH7.4) was replaced with fresh medium at different pH levels(pH6.5, pH6.0, pH5.5 and pH5.0) and/or in the presence of mannitol orglucose (10 and 100 mM) at a specific release time (5 h). Values werereported as the means for each duplicate samples.

PTX release from NCM was rapid with almost 30% of PTX released withinthe first 9 h independently from the pH of the release medium or thepresence of diols. PTX release from BCM3 crosslinked via phenyl boronatewas significantly slower than NCM but faster than BCM4 with nitro-phenylboronate crosslinking at pH7.4. PTX release from BCM3 was promoted whendecreasing the pH of the medium from 7.4 to 6.5 while that of BCM4 wasaccelerated at pH 5.5. In the presence of glucose at its physiologicallevel (2-10 mM) or even higher concentration (50 mM), PTX release fromBCM3 and BCM4 was similar to that in the release media without glucose.It was noted that PTX release was not sensitive to 10 mM mannitol butcould be gradually facilitated as the concentration of mannitolincreased up to the range of 50-100 mM.

In order to simulate the in vivo situations, the PTX release from BCM4was first incubated under psychological pH for a period of time (e.g. 5hr) and then was triggered with acidic pH and/or mannitol. The PTXrelease from BCM4 was significantly slower than that from NCMs at theinitial 5 h. When 100 mM mannitol was added or the pH of the medium wasadjusted to 5.0 at the 5 hr time point, there was a burst of drugrelease from the BCM4. It should be noted that the PTX release can befurther accelerated via the combination of 100 mM of mannitol and pH5.0. This two-stage release strategy can be exploited so that prematuredrug release can be minimized during circulation in vivo followed byrapid drug release triggered by the acidic tumor microenvironment, orupon micelle exposure to the acidic compartments of cancer cells or bythe additional administration of mannitol.

Example 16 Treatment of Asthma

To evaluate the therapeutic efficacy of micelle-encapsulateddexamethasone, we use an asthma mouse model that employs an ovalbumin(OVA) in alum (Ova/alum) sensitization and OVA aerosol exposure regimen.This mimics the pathological features of asthma in terms of T cell andeosinophil driven-inflammatory and mucous hypersecretory responses andexhibits the structural airway changes of chronic asthma (ref 1 & 2).This experiment was done in collaboration with Dr. Nick Kenyon of UCDavis. Dexamethasone was nanoformulated with PEG^(5k)-CA⁸ andPEG^(2k)-CA₄. OVA exposed mice were treated with dexamethasone-loadednanoparticles, dexamethasone alone, or PBS. Both dexamethasonenanoformulations decreased lung lavage cell counts and eosinophil countsmore than Dex alone (FIG. 34).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference. Where a conflictexists between the instant application and a reference provided herein,the instant application shall dominate.

1. A compound of formula I:(PEG)_(m)-A(Y¹)_(p)-L-D(Y²)_(q)—(R)_(n)  (I) wherein A is linked to atleast one PEG group; D is a dendritic polymer having a single focalpoint group, a plurality of branched monomer units X and a plurality ofend groups; L is a bond or a linker linked to the focal point group ofthe dendritic polymer; each PEG is a polyethyleneglycol (PEG) polymer,wherein each PEG polymer has a molecular weight of 1-100 kDa; each R isindependently selected from the group consisting of the end group of thedendritic polymer, a hydrophobic group, a hydrophilic group, anamphiphilic compound and a drug, such that when R is not an end groupthen each R is linked to one of the end groups; each Y¹ and Y² is acrosslinkable group independently selected from the group consisting ofboronic acid, dihydroxybenzene and a thiol; subscript m is an integerfrom 0 to 20; subscript n is an integer from 2 to 20, wherein subscriptn is equal to the number of end groups on the dendritic polymer, andwherein at least half the number n of R groups are each independentlyselected from the group consisting of a hydrophobic group, a hydrophilicgroup, an amphiphilic compound and a drug; and each of subscripts p andq are 0 or from 2 to 8, such that one of subscripts p and q is from 2 to8.
 2. The compound of claim 1, wherein each branched monomer unit X isindependently selected from the group consisting of a diamino carboxylicacid, a dihydroxy carboxylic acid and a hydroxylamino carboxylic acid.3. The compound of claim 2, wherein each diamino carboxylic acid isindependently selected from the group consisting of 2,3-diaminopropanoic acid, 2,4-diaminobutanoic acid, 2,5-diaminopentanoic acid(ornithine), 2,6-diaminohexanoic acid (lysine), (2-Aminoethyl)-cysteine,3-amino-2-aminomethyl propanoic acid, 3-amino-2-aminomethyl-2-methylpropanoic acid, 4-amino-2-(2-aminoethyl)butyric acid and5-amino-2-(3-aminopropyl)pentanoic acid.
 4. The compound of claim 2,wherein each dihydroxy carboxylic acid is independently selected fromthe group consisting of glyceric acid, 2,4-dihydroxybutyric acid,2,2-Bis(hydroxymethyl)propionic acid, 2,2-Bis(hydroxymethyl)butyricacid, serine and threonine.
 5. The compound of claim 2, wherein eachhydroxylamino carboxylic acid is independently selected from the groupconsisting of serine and homoserine.
 6. The compound of claim 2, whereinthe diamino carboxylic acid is an amino acid.
 7. The compound of claim2, wherein each branched monomer unit X is lysine.
 8. The compound ofclaim 1, wherein each R is independently selected from the groupconsisting of cholic acid, (3α, 5β, 7α,12α)-7,12-dihydroxy-3-(2,3-dihydroxy-1-propoxy)-cholic acid (CA-4OH),(3α, 5β, 7α, 12α)-7-hydroxy-3,12-di(2,3-dihydroxy-1-propoxy)-cholic acid(CA-5OH), (3α, 5β, 7α,12α)-7,12-dihydroxy-3-(3-amino-2-hydroxy-1-propoxy)-cholic acid(CA-3OH—NH₂), cholesterol formate, doxorubicin, and rhein.
 9. Thecompound of claim 1, wherein each R is cholic acid.
 10. The compound ofclaim 1, wherein linker L is selected from the group consisting ofpolyethylene glycol, polyserine, polyglycine, poly(serine-glycine),aliphatic amino acids, 6-amino hexanoic acid, 5-amino pentanoic acid,4-amino butanoic acid and beta-alanine.
 11. The compound of claim 1,wherein linker L is the Ebes linker having the formula:


12. (canceled)
 13. The compound of claim 1, having the structure:PEG-A(Y¹)_(p)-D-(R)_(n)  (Ia) wherein subscript p is an integer from 2to 8 and subscript q is
 0. 14. The compound of claim 1, having thestructure selected from the group consisting of:

wherein each L′ is a linker Ebes; PEG is PEG5k; each R is cholic acid;each branched monomer unit X is lysine; and Y¹ is selected from thegroup consisting of carboxyphenylboronic acid, carboxynitrophenylboronic acid and 3,4-dihydroxybenzoic acid.
 15. The compound of claim 1,having the structure:PEG-A-D(Y²)_(q)—(R)_(n)  (Ib) wherein subscript p is 0 and subscript qis an integer from 2 to
 8. 16. The compound of claim 1, having thestructure:

wherein A is lysine; each L′ is a linker Ebes; PEG is PEG5k; each R ischolic acid; each branched monomer unit X is lysine; and each Y² iscysteine.
 17. A reversibly crosslinked nanocarrier having an interiorand an exterior, the nanocarrier comprising at least two conjugates,wherein each conjugate comprises: a polyethylene glycol (PEG) polymer;at least two amphiphilic compounds having both a hydrophilic face and ahydrophobic face; at least two crosslinking groups; and a dendriticpolymer covalently attached to the PEG, the amphiphilic compounds, andthe crosslinking groups, wherein each conjugate self-assembles in anaqueous solvent to form the nanocarrier such that a hydrophobic pocketis formed in the interior of the nanocarrier by the orientation of thehydrophobic face of each amphiphilic compound towards each other,wherein the PEG of each conjugate self-assembles on the exterior of thenanocarrier, and wherein at least two conjugates are reversiblycrosslinked via the crosslinking groups.
 18. The nanocarrier of claim17, wherein each conjugate comprises a compound of claim
 1. 19. Thenanocarrier of claim 17, wherein the crosslinking groups are selectedfrom the group consisting of thiol, boronic acid and dihydroxybenzene.20.-25. (canceled)
 26. The nanocarrier of claim 17, wherein eachamphiphilic compound is cholic acid.
 27. A method of delivering a drugto a subject in need thereof, comprising: administering a nanocarrier ofclaim 18 to the subject, wherein the nanocarrier comprises the drug anda plurality of cross-linked bonds; and cleaving the cross-linked bondsusing a bond cleavage component, such that the drug is released from thenanocarrier, thereby delivering the drug to the subject. 28.-29.(canceled)
 30. A method of reversing the cross-linking of the reversiblycrosslinked nanocarrier of claim 17, comprising contacting thereversibly crosslinked nanocarrier with a bond cleavage componentsuitable for cleaving the cross-linked bond, thereby reversing thecross-linking of the reversibly crosslinked nanocarrier. 31.-34.(canceled)
 35. A method of treating a disease, comprising administeringto a subject in need of such treatment, a therapeutically effectiveamount of a nanocarrier of claim 18, wherein the nanocarrier furthercomprises a drug. 36.-39. (canceled)